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Effects of Cardioactive Peptides on Myocardial cAMP Levels in the Snail Helix aspersa
Peptides, Vol. 18, No. 3, pp. 355–360, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/97 $17.00 / .00
PII S0196-9781(96)00335-X
Effects of Cardioactive Peptides on Myocardial cAMP Levels in the Snail Helix aspersa GERHARD REICH,1 KAREN E. DOBLE, DAVID A. PRICE AND MICHAEL J. GREENBERG The Whitney Laboratory, University of Florida, St. Augustine, FL 32086 Received 13 September 1996; Accepted 10 December 1996 REICH, G., K. E. DOBLE, D. A. PRICE AND M. J. GREENBERG. Effects of cardioactive peptides on myocardial cAMP levels in the snail Helix aspersa. PEPTIDES 18 (3) 355–360, 1997.—Several cardioactive peptides from the pulmonate snail Helix aspersa were tested for their effects on myocardial cAMP levels, but only the family of small cardioactive peptides (SCPs) were clearly effective. SCP increased cAMP in a dose dependent manner; the time course was phasic. The structure-activity relations of this effect were examined with a set of 3 synthetic analogs having characteristics, at the carboxyterminal, of both the SCPs and FMRFamide-related peptides. The adenylate cyclase activator forskolin mimicked the mechanical effect of SCPs on the heartbeat. We conclude that the effect of SCPs on the Helix heart may be mediated by cAMP. q 1997 Elsevier Science Inc. FMRFamide
SCP
cAMP
Helix aspersa
Cardioactive peptides
THE heart of the pulmonate snail Helix aspersa consists of two chambers: an atrium that receives haemolymph from the lung, and a ventricle that exits to the aorta; back flow is prevented by valves. The myogenic heart is innervated by two branches of the visceral nerve trunk; one enters the atrium, the other innervates ventricle and aorta (9). To date, more than ten cardioactive peptides, distributed in two families, have been identified in Helix aspersa : a pair of small cardioactive peptides (SCPs) and several FMRFamide-related peptides (FaRPs) (9,21). The FaRPs comprise two subgroups encoded on separate precursors: the tetrapeptides and heptapeptides (15). Most of these peptides, when assayed on isolated ventricles, are excitatory; only one, pQDPFLRIamide, which occurs in a single copy on the heptapeptide precursor (15), decreases the systolic amplitude (9). The excitatory peptides have distinctly different potencies: the SCPs are the most potent; the FMRFamide-related heptapeptides are intermediate in activity; and the tetrapeptides FMRFamide and FLRFamide are the least potent (21). The SCPs and both groups of FaRPs have been detected in the brain, the visceral nerve trunk and the aorta, but extracts of ventricles only contain the tetrapeptides and SCPs (9); the heptapeptides are lacking. We presume that cardioactive peptides other than those detected to date are involved in cardiac regulation in Helix aspersa . In particular, 63 different bioactive peptides from 7 peptide families have been identified in the closely related snail Helix pomatia by Y. Muneoka and coworkers (8,16). In preliminary experiments we have found that at least one of these peptides, APGWamide, is also cardioactive.
We are interested in the set of mechanisms by which this array of structurally different effector molecules modulates the heart. We have therefore assayed a set of endogenous cardioactive molecules for their effect on cAMP levels in isolated ventricles: FMRFamide, related heptapeptides, SCPb, 5HT and APGWamide. METHOD
Animals and Hearts Helix aspersa were collected from the gardens of Fullerton, California; picked and then shipped to the Whitney Laboratory by Robert A. Koch. In our lab, they were transferred into plastic containers and maintained in aestivation. At least 3 days before an experiment, the snails were first activated with an increase in humidity and then fed lettuce. The entire heart — atrium and ventricle — was dissected from each animal and placed in a Helix saline of the following composition: 80 mM NaCl, 20 mM HEPES, 7 mM CaCl2, 5 mM KCl and 5 mM MgCl2 (pH 7.5). The tissue was washed with this saline, every 15 min for 2 h at room temperature. In preliminary experiments the hearts were cut into several pieces, and each was tested individually for its cAMP response; no differences between regions of the heart were detected. Peptides and Chemicals FMRFamide, SCPb (MNYLAFPRMamide), forskolin, 2 *-Omonosuccinyladenosine 3 *:5 *cyclic monophosphate tyrosyl methyl ester, and 3-isobutyl-1-methyl-xanthin (IBMX) were purchased from Sigma (St. Louis, USA); pQDPFLRIamide and
1 Requests for reprints should be addressed to Gerhard Reich, The Whitney Laboratory, University of Florida, 9505 Ocean Shore Blvd., St. Augustine, FL 32086-8623; Fax: (904)461-4008; E-mail: [email protected]
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APGWamide were synthesized by Research Genetics (Huntsville, Alabama, USA); and pQDPFLRFamide was from Peninsula Laboratories Inc. The synthetic analogs FPRMamide, FPRFamide, and FMRMamide were synthesized by the Protein Core Facility of the University of Florida Interdisciplinary Center for Biotechnology Research. In our laboratory, these peptides were deprotected and cleaved from the resin with trifluoromethanesulfonic acid, according to the Applied Biosystems protocol. The resulting samples were purified by HPLC and quantified by automated amino acid analysis (Hitachi model 835). The cAMP antiserum used for RIA was a gift from H.-Y. T. Yang (NIMH, Neuroscience Center at St. Elizabeth’s, Washington, DC). cAMP Assay The hearts were stimulated by the addition of peptide dissolved in Helix saline. The adenylate cyclase activator forskolin was used as a nonspecific control stimulant. In some experiments, the tissue was incubated with phosphodiesterase inhibitors (1 mM theophylline, 0.1 mM IBMX in saline) for 5 min prior to stimulation. After a given time, we stopped the stimulation by shock-freezing the hearts on an aluminum block immersed in liquid nitrogen. cAMP was then extracted from the tissue (30 min at 707C) with ethanol containing 10 mM theophylline and 2.5 mM IBMX. The time course of the cAMP response was determined by varying the incubation times in steps, from 2 s to 12 min. Controls without peptide were performed for each incubation time. The peptide concentration was usually 10 05 M, but some experiments were also performed with lower doses. The experiments were usually carried out at room temperature, but some additional studies of the time course of the response were done at 07C. To prepare dose-response curves, the peptides were applied in concentrations from 10 08 M to 10 05M; the incubation time chosen for these experiments was 1.5 min. cAMP Radioimmunoassay The cAMP extracted from the tissue was measured by RIA. As trace we used a cAMP derivative that was iodinated with 125I by a chloramine T method as follows: About 1 nmol of 2 *O-monosuccinyladenosine 3 *:5 *cyclic monophosphate tyrosyl methyl ester in 10 ml 0.5 M phosphate buffer (pH 7) was combined with radioactive sodium iodide solution (1.5 mCi in 1-3 ml). Chloramine-T solution (5 ml of 2 mg/ml in 0.5M phosphate buffer) was added, and the solution was mixed for 30 s. Sodium metabisulfite (100 ml of 5 mg/ml in phosphate buffer) was added. After sitting for 5 min, the mixture was applied to a conditioned Sep-Pak cartridge (C18, Waters Associates) which was then washed with 20 ml of water, and the radiolabeled compound was eluted with 80% aqueous acetonitrile containing 0.05% TFA. A total of 5 ml was collected in 1 ml fractions. Usually the first fraction, containing the highest amount of radioactivity, was stored at 0207C and used for the RIA. The antiserum was applied in a final dilution of 1:21,000. The RIA buffer consisted of: 0.2M Tris, 0.06% dextran, 0.1% BSA, 10 mM EDTA, and 10 mM theophylline, pH 7.4. An aliquot (10– 50 ml) of the ethanol extracts was dried and redissolved in RIA buffer. The cAMP was acetylated with 5 ml acetic anhydride/ triethylamine (1:2 v/v) per 50 ml sample. The trace (10,000 cpm/ tube) and antiserum were added, and incubation was carried out overnight at 47C. Bound and free trace were separated by the addition of 1% BSA in RIA buffer, followed by precipitation of
the protein with ice cold ethanol for 1 h at 47C. After centrifugation, the supernatant was discarded, and the protein pellet was counted in a gamma counter. The hearts were blotted between paper tissues and weighed, and the cAMP values were related to those weights. The control levels of cAMP varied from one experiment to the next, ranging from 1 to 8 pmol/mg tissue. Therefore the data from any experiment were normalized with respect to the mean of the controls from that experiment. The t-test for independent samples was used to compare the cAMP levels of treated and untreated hearts; the p value was õ 0.01. Mechanical Recordings Whole Helix hearts were excised, a canula was inserted through the atrium into the ventricle, and this preparation was mounted in a setup similar to that described by Payza (19). The heart was perfused with saline which was delivered from one of 2 reservoirs (control and test) that were connected through a three-way valve to the canula. In contrast to previous experiments (19) the test agent was not delivered in a relatively small bolus. Rather, the hearts were perfused for up to 10 min with a solution containing peptide or forskolin, and this was done by merely turning the valve to the appropriate perfusion fluid. This procedure allowed us to compare the mechanical response and the change in cAMP level over the same time course. The reservoirs were mounted so that the height of the water column would be kept constant, precluding pressure artifacts. The mechanical activity of the myocardium was recorded with a force transducer which was attached to the aorta and connected to a polygraph (Grass model 79C). RESULTS
Effects of Cardioactive Agents on cAMP Levels Several cardioactive peptides and 5HT (all at 10 05 M) were tested for their effects on the cAMP levels in isolated ventricles. Forskolin (10 05 M), which activates adenylate cyclase directly, was also applied as a positive control (Fig. 1). SCPb increased cAMP levels markedly over control levels. In contrast to this robust response, that of 5HT was only modest, and no detectable effects were observed after stimulation with FMRFamide, the excitatory heptapeptide pQDPFLRFamide, or the inhibitory peptides pQDPFLRIamide and APGWamide. Since SCPb potently increases cAMP levels whereas FMRFamide is inactive, we tested the effects of three additional synthetic analogs: FPRMamide (the C-terminal tetrapeptide of SCPb), FPRFamide and FMRMamide. The latter two peptides are intermediate in structure between the C-terminals of the SCPs and FMRFamide. Of these analogs, only the two containing proline — FPRMamide and FPRFamide — significantly increased cAMP levels over controls (5.4- and 2-fold, respectively), and neither analog was nearly as active as SCPb. The very small increase produced by FMRMamide was not significantly different from controls (Fig. 2). Characterization of the SCPb Effect The time course of the increase in cAMP levels was distinctive. At room temperature, the response to SCPb was already maximal within 1-2 min, but this increase was followed by a fast decrease in cAMP levels which approached their control values asymptotically (Fig. 3). The control level of cAMP remained constant over the 12 minute duration of the experiment (not shown).
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SCPs RAISE cAMP IN SNAIL MYOCARDIUM
FIG. 1. Effect of various cardioactive peptides, 5HT, and forskolin on myocardial cAMP. Each drug was applied for 1.5 min, except that forskolin was applied for 10 min. All drugs were used at a concentration of 10 05 M. Error bars Å SEM; * Å p õ 0.01. n Å 17,31 (SCPb, control); 6,19 (FMRFamide, control); 12,12 (pQDPFLRFamide, control); 8,8 (pQDPFLRIamide, control); 9,8 (APGWa, control); 7,15 (5HT, control); 10,8 (forskolin, control).
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FIG. 3. Time courses at 07C and room temperature for the stimulation of cAMP levels by SCPb (10 05 M). Error bars Å SEM. n Å 11 for room temperature; n Å 6 for 07C.
When the experiment was carried out at 07C, the time course was markedly different. The initial increase was slower, and the elevation of cAMP was lower; a maximal 7-fold elevation was measured after 6 min. The effect of the lowered temperature, in essence, was to keep the levels of cAMP elevated for a longer period. The effect of SCPb on cAMP levels was clearly related to dose (Fig. 4). Threshold was 10 08 M, and the maximal effect (an 8-15 fold increase over control levels) was produced by concentrations of 10 06 –10 05M. Application of phosphodiesterase inhibitors altered neither the threshold nor the relative shape of the time course at room temperature; but the maximal increase in cAMP was 5 to 6 times greater than without inhibitors (not shown). Mechanical Activity
FIG. 2. Structure-activity relations of the effect of SCPb on cAMP levels. FPRMa is the C-terminal tetrapeptide of SCPb; FMRMa and FPRFa are intermediates, each with 1 substitution. aÅamide. Doses were 10 05 M. Error bars Å SEM; * Å p õ 0.01. n Å 11,25 (FPRMa, control); 8,18 (FPRFa, control); 8,18 (FMRMa, control). Data for SCPb and FMRFamide are from Fig.1.
Ventricles were perfused with SCPb and forskolin, and the mechanical activity was recorded. In the past, drugs were injected as a bolus into the perfusion stream, so the effect lasted only for seconds. But because the increase in cAMP has a time course lasting minutes, we perfused the ventricles with test solutions for a longer period. Under these conditions, isolated Helix hearts respond to SCPb, as usual, with an increase of the systolic amplitude and a decrease of the diastolic tone; moreover the mechanical response persists for as long as SCPb is present (Fig. 5). Threshold for a mechanical activation by SCPb was 10 011 M, but individual differences occurred. At doses of peptide higher than 10 010 M the heartbeat became arrhythmic after 0.51 min of perfusion. In summary, the augmented mechanical cardioactivity caused by SCPb at room temperature is distinct — in both time course and threshold — from the phasic elevation of cAMP produced in isolated hearts by the same peptide.
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FIG. 4. Dose-response relationship of the SCPb effect on cAMP levels. Incubation time with peptide was 1.5 min at room temperature. n Å 15; error bars Å SEM.
Treatment of isolated hearts with forskolin (10 05M) produced an effect similar to that of SCPb; systolic amplitude increased and diastolic tone declined. But the cardioexcitation evoked by forskolin did differ from the SCPb effects in that it outlasted the actual perfusion time by more than 10 min. APGWamide inhibits the beat of isolated Helix ventricles at a threshold of 10 08 M to 2 1 10 08 M by decreasing both the amplitude and the diastolic tone (Fig. 6). A slight increase in dose to 4 1 10 08 M led to a complete cardiac arrest. After 5 min of washout, the hearts recovered and continued to beat spontaneously. DISCUSSION
The effects of cardioexcitatory peptides on the heartbeat of the snail are very similar; the mechanical responses are distinguishable only by differences in relative potency or in pattern of beat. In contrast — as this report shows — the intracellular effects of these same peptides are markedly different. In particular, although SCPb causes a robust increase in myocardial cAMP, other cardioexcitatory peptides lack this effect and, therefore, probably activate different second messenger pathways in the Helix myocardium. Among the cardioexcitatory peptides that have been identified in Helix, the SCPs are the most potent mechanical stimulators (21), and they also increase myocardial cAMP strongly. Since elevation of myocardial cAMP with forskolin is accompanied by an increase in mechanical activity similar to that caused by the peptide, the SCP-induced cardioexcitation is likely to be mediated by cAMP. The thresholds and the time courses of the mechanical stimulation and cAMP elevation are different, but the activation of adenylate cyclase through a G-protein linked receptor is only the first step in augmenting cardioactivity. The final response of the beating heart to SCPb may therefore be amplified, so that a dose
FIG. 5. Mechanical responses of Helix ventricles to SCPb and forskolin (auxotonic recording). The drugs were applied over 10 min. (period indicated by horizontal bar). Doses are concentrations in the perfusing saline.
higher than 10 010 M already leads to arrythmicity, whereas changes in cAMP cannot be detected biochemically at that dose. We speculate that, in the animal, the peptide is released in short bursts, so that arrhythmia is not established, even at higher local concentrations. The more sustained elevation of cAMP levels at lower temperatures might reflect a longer biological half life of the peptide agonist, or a decreased activity of phosphodiesterases. The effect of temperature on the amplitude of the cAMP increase is complex and cannot be addressed here. Although the effects of purified extracts on isolated Helix hearts provided the initial evidence for the existence of ‘‘small cardioactive peptides’’ (11,12), the actual peptides were first purified and sequenced from Aplysia californica (14,17), an opisthobranch mollusc. The physiology of the SCPs have been studied intensively in Aplysia: in the heart (13) and more recently in the accessory radula closer (ARC) muscle (4). Moreover, the excitatory effects on both tissues is mediated by cAMP. The cas-
FIG. 6. Mechanical response of Helix ventricles to APGWamide. Period of application of the peptide is indicated by bars. Dose is the concentration in the saline perfusing the heart.
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cade in the ARC muscle includes the activation of a cAMP-dependent kinase and the phosphorylation of the 750 kDa protein twitchin (22). Thus, the modulation of muscle activity by SCPs may be mediated by a cAMP pathway in all of the molluscan tissues that have been studied. In sharp contrast to the robust effect of the SCPs on cAMP levels, the effects of the FaRPs - both tetrapeptides and heptapeptides - were indistinguishable from the controls. This marked difference in the actions of the FaRPs and SCPs could not have been anticipated. Not only are the two types of peptides cardioexcitatory, they also share the C-terminal sequence (F-x1-R-x2-amide, where x1 and x2 are hydrophobic residues) and, on this basis, were even considered to have some familial relationship (17). More to the point, however, FMRFamide has been shown to increase cAMP levels in some other molluscan preparations. For example, the heart of the freshwater clam Lampsilis claibornensis is inhibited by FMRFamide (18), that of the estuarine clam Mercenaria mercenaria is excited (7), and in both cases the effect is accompanied by an increase of myocardial cAMP. And FMRFamide also activates adenylate cyclase in membrane preparations of squid optic lobes (3). Our finding that the synthetic tetrapeptides intermediate in structure between an SCP (-FPRMamide) and a FaRP (-FMRFamide) C-terminal, are also intermediate in their effects on cAMP levels suggests that the apparent structural similarities in the two families lack functional significance. The potency of the analogs in increasing myocardial cAMP is correlated with their activity on the beat of isolated snail ventricles: i.e., the proline containing analogs were 15 times (FPRMamide) and 5 times (FPRFamide) more potent cardioexcitors than FMRFamide (6). At present, the effects of FMRFamide and related peptides cannot be ascribed to any one signal transduction pathway within molluscs, because a variety of mechanisms - in addition to the increase in cAMP - have been observed in different molluscan preparations. For example, the excitatory effect of FMRFamide
on the heart of the marsh mussel Geukensia seems to involve a decrease of IP3 (1), but a small increase of IP3 seems to accompany the effect of FMRFamide on the tentacle retractor muscle of Helix aspersa (5). In Aplysia sensory neurons, FMRFamide was reported to activate a phospholipase A2 pathway with metabolites of arachidonic acid as second messengers (2). Finally, a FMRFamide gated sodium channel has recently been cloned from a Helix aspersa cDNA library of the nervous system, but expression of this channel was not observed in the heart (10). In Helix aspersa , the tetrapeptide and heptapeptide FaRPs can be distinguished by their bioactivity (21) and by their behavior in radioligand-receptor binding assays. Thus, the radioligand dYFnLRFamide was specifically displaced from Helix heart and brain membranes by the tetrapeptide FaRPs, but not by heptapeptides (19,20). The two distinct sets of molluscan FaRPs must therefore have different receptors, and their effects might well be mediated by different transduction pathways. The inhibitory effect of APGWamide on the beat of isolated ventricles indicates that a mechanism antagonistic to the excitatory peptides might be present. But a cardioregulatory role for APGWamide in Helix aspersa has not been established, and the actual peptide, though present in Helix pomatia (16), has yet to be identified in H. aspersa. In conclusion, the SCPs are the only peptides of the known cardioactive blend in Helix aspersa that increase the levels of cAMP; the second messengers of the other peptides remain unknown. ACKNOWLEDGEMENTS
This work was supported by NIH grant HL28440 to M.J. Greenberg and DFG grant Re1/1 to G. Reich. We are indebted to Dr. H.-Y.T.Yang for her generous gift of the cAMP antiserum. This is contribution number 316 from the Tallahassee, Sopchoppy & Gulf Coast Marine Biological Association.
REFERENCES 1. Bayakly, N. A.; Deaton, L. E. The effects of FMRFamide, 5-hydroxytryptamine and phorbol esters on the heart of the mussel Geukensia demissa. J. Comp. Physiol. B 162:463–468; 1992. 2. Piomelli, D.; Volterra, A.; Dale, N.; Siegelbaum, S. A.; Kandel, E. R.; Schwartz, J. H; Belardetti, F. Lipoxygenase metabolites of arachidonic acid as second messengers for presynaptic inhibition of Aplysia sensory cells. Nature 328:38–43; 1987. 3. Chin, G. J.; Payza, K.; Price, D. A.; Greenberg, M. J. Doble, K. E. Characterization and solubilization of the FMRFamide receptor of squid. Biol. Bull. 187:185–199; 1994. 4. Cropper, E. C.; Price, D. A.; Tenenbaum, R.; Kupfermann, I. Weiss, K. R. Release of peptide cotransmitters from a cholinergic motor neuron under physiological conditions. Proc. Natl. Acad. Sci. USA 87:933–937; 1990. 5. Falconer, S. P. W, Carte, A. N.; Downes, C. P. Cottrell, G. A. The neuropeptide Phe-Met-Arg-Phe-NH2 increases levels of inositol 1,4,5-triphosphate in the tentacle retractor muscle of Helix aspersa. Exp. Physiol. 78:757–766; 1993. 6. Greenberg, M. J.; Doble, K. E.; Lesser, W.; Dunn, B. M. Price, D. A. The structure-activity relations of FMRFamide depend on the assay used to measure them. Soc. Neurosci. Abstr. 19:929; 1993. 7. Higgins, W. J.; Price, D. A. Greenberg, M. J. FMRFamide increases the adenylate cyclase activity and cyclic AMP level of molluscan heart. Eur. J. Pharm. 48:425–430; 1978. 8. Ikeda, T.; Minakata, H.; Fujita, T.; Muneoka, Y.; Kiss, T.; Hiripi, L. Nomoto, K. Neuropeptides isolated from Helix pomatia part 1. Peptides related to MIP, buccalin, myomodulin-CARP and SCP. In: Yanihara, N., Ed. Peptide chemistry. ESCOM, Leiden; 1992:576– 578.
9. Lesser, W. Greenberg, M. J. Cardioactive regulation by endogenous small cardioactive peptides and FMRFamide-related peptides in the snail Helix aspersa . J. Exp. Biol. 178:205 – 230; 1993. 10. Lingueglia, E.; Champigny, G.; Lazdunski, M. Barbry, P. Cloning of the amiloride sensitive FMRFamide peptide-gated sodium channel. Nature 378:730–733; 1995. 11. Lloyd, P. E. Distribution and molecular characteristics of cardioactive peptides in the snail, Helix aspersa. J. Comp. Physiol. A 128:269–276; 1978. 12. Lloyd, P. E. Biochemical and pharmacological analyses of endogenous cardioactive peptides in the snail, Helix aspersa. J. Comp. Physiol. A 138:265–270; 1980. 13. Lloyd, P. E.; Kupfermann I. Weiss K. R. Two endogenous neuropeptides (SCPA and SCPB ) produce a cAMP-mediated stimulation of cardiac activity in Aplysia. J. Comp. Physiol. A 156:659–667; 1985. 14. Lloyd, P. E.; Kupfermann, I. Weiss, K. R. Sequence of small cardioactive peptide A: A second member of a class of neuropeptides in Aplysia. Peptides 8:179–184; 1987. 15. Lutz, E. M.; Macdonald, M.; Hettle, S.; Price, D. A.; Cottrell, G. A. Sommerville, J. Structure of cDNA clones and genomic DNA encoding FMRFamide-related peptides (FaRPs) in Helix. Mol. Cell. Neurosci. 3:373–382; 1992. 16. Minakata, H.; Ikeda, T.; Fujita, T.; Kiss, T.; Hiripi, L. Muneoka, Y. Nomoto, K. Neuropeptides isolated from Helix pomatia part 2. FMRFamide-related peptides, S-Iamide peptides, FR peptides and others. In Peptide Chemistry. Yanihara, N. Ed., ESCOM, Leiden; 1992:579–582;.
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17. Morris, H. R.; Panico, M.; Karplus, A.; Lloyd, P. E. Riniker, B. Elucidation by FAB-MS of the structure of a new cardioactive peptide from Aplysia. Nature 300:643–645; 1982. 18. Painter, S. D. FMRFamide inhibition of a molluscan heart is accompanied by increases in cyclic AMP. Neuropeptides 3:19 – 27; 1982. 19. Payza, K. FMRFamide receptors in Helix aspersa. Peptides 8:1065– 1074; 1987. 20. Payza, K.; Greenberg, M. J. Price, D. A. Further characterization of Helix FMRFamide receptors: Kinetics, tissue distribution, and in-
teractions with the endogenous heptapeptides. Peptides 10:657–661; 1988. 21. Price, D. A.; Lesser, W.; Lee, T. D.; Doble, K. E.; Greenberg, M. J. Seven FMRFamide-related and two SCP-related cardioactive peptides from Helix. J. Exp. Biol. 154:421–437; 1990. 22. Probst, W. C.; Cropper, E. C.; Heierhorst, J.; Hooper, S. L.; Jaffe, H.; Vilim, F.; Beushausen, S.; Kupfermann, I. Weiss, K. R. cAMPdependent phosphorylation of Aplysia twitchin may mediate modulation of muscle contractions by neuropeptide cotransmitters. Proc. Natl. Acad. Sci. USA 91:8487–8491; 1994.
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Effects of Cardioactive Peptides on Myocardial cAMP Levels in the Snail Helix aspersa GERHARD REICH,1 KAREN E. DOBLE, DAVID A. PRICE AND MICHAEL J. GREENBERG The Whitney Laboratory, University of Florida, St. Augustine, FL 32086 Received 13 September 1996; Accepted 10 December 1996 REICH, G., K. E. DOBLE, D. A. PRICE AND M. J. GREENBERG. Effects of cardioactive peptides on myocardial cAMP levels in the snail Helix aspersa. PEPTIDES 18 (3) 355–360, 1997.—Several cardioactive peptides from the pulmonate snail Helix aspersa were tested for their effects on myocardial cAMP levels, but only the family of small cardioactive peptides (SCPs) were clearly effective. SCP increased cAMP in a dose dependent manner; the time course was phasic. The structure-activity relations of this effect were examined with a set of 3 synthetic analogs having characteristics, at the carboxyterminal, of both the SCPs and FMRFamide-related peptides. The adenylate cyclase activator forskolin mimicked the mechanical effect of SCPs on the heartbeat. We conclude that the effect of SCPs on the Helix heart may be mediated by cAMP. q 1997 Elsevier Science Inc. FMRFamide
SCP
cAMP
Helix aspersa
Cardioactive peptides
THE heart of the pulmonate snail Helix aspersa consists of two chambers: an atrium that receives haemolymph from the lung, and a ventricle that exits to the aorta; back flow is prevented by valves. The myogenic heart is innervated by two branches of the visceral nerve trunk; one enters the atrium, the other innervates ventricle and aorta (9). To date, more than ten cardioactive peptides, distributed in two families, have been identified in Helix aspersa : a pair of small cardioactive peptides (SCPs) and several FMRFamide-related peptides (FaRPs) (9,21). The FaRPs comprise two subgroups encoded on separate precursors: the tetrapeptides and heptapeptides (15). Most of these peptides, when assayed on isolated ventricles, are excitatory; only one, pQDPFLRIamide, which occurs in a single copy on the heptapeptide precursor (15), decreases the systolic amplitude (9). The excitatory peptides have distinctly different potencies: the SCPs are the most potent; the FMRFamide-related heptapeptides are intermediate in activity; and the tetrapeptides FMRFamide and FLRFamide are the least potent (21). The SCPs and both groups of FaRPs have been detected in the brain, the visceral nerve trunk and the aorta, but extracts of ventricles only contain the tetrapeptides and SCPs (9); the heptapeptides are lacking. We presume that cardioactive peptides other than those detected to date are involved in cardiac regulation in Helix aspersa . In particular, 63 different bioactive peptides from 7 peptide families have been identified in the closely related snail Helix pomatia by Y. Muneoka and coworkers (8,16). In preliminary experiments we have found that at least one of these peptides, APGWamide, is also cardioactive.
We are interested in the set of mechanisms by which this array of structurally different effector molecules modulates the heart. We have therefore assayed a set of endogenous cardioactive molecules for their effect on cAMP levels in isolated ventricles: FMRFamide, related heptapeptides, SCPb, 5HT and APGWamide. METHOD
Animals and Hearts Helix aspersa were collected from the gardens of Fullerton, California; picked and then shipped to the Whitney Laboratory by Robert A. Koch. In our lab, they were transferred into plastic containers and maintained in aestivation. At least 3 days before an experiment, the snails were first activated with an increase in humidity and then fed lettuce. The entire heart — atrium and ventricle — was dissected from each animal and placed in a Helix saline of the following composition: 80 mM NaCl, 20 mM HEPES, 7 mM CaCl2, 5 mM KCl and 5 mM MgCl2 (pH 7.5). The tissue was washed with this saline, every 15 min for 2 h at room temperature. In preliminary experiments the hearts were cut into several pieces, and each was tested individually for its cAMP response; no differences between regions of the heart were detected. Peptides and Chemicals FMRFamide, SCPb (MNYLAFPRMamide), forskolin, 2 *-Omonosuccinyladenosine 3 *:5 *cyclic monophosphate tyrosyl methyl ester, and 3-isobutyl-1-methyl-xanthin (IBMX) were purchased from Sigma (St. Louis, USA); pQDPFLRIamide and
1 Requests for reprints should be addressed to Gerhard Reich, The Whitney Laboratory, University of Florida, 9505 Ocean Shore Blvd., St. Augustine, FL 32086-8623; Fax: (904)461-4008; E-mail: [email protected]
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APGWamide were synthesized by Research Genetics (Huntsville, Alabama, USA); and pQDPFLRFamide was from Peninsula Laboratories Inc. The synthetic analogs FPRMamide, FPRFamide, and FMRMamide were synthesized by the Protein Core Facility of the University of Florida Interdisciplinary Center for Biotechnology Research. In our laboratory, these peptides were deprotected and cleaved from the resin with trifluoromethanesulfonic acid, according to the Applied Biosystems protocol. The resulting samples were purified by HPLC and quantified by automated amino acid analysis (Hitachi model 835). The cAMP antiserum used for RIA was a gift from H.-Y. T. Yang (NIMH, Neuroscience Center at St. Elizabeth’s, Washington, DC). cAMP Assay The hearts were stimulated by the addition of peptide dissolved in Helix saline. The adenylate cyclase activator forskolin was used as a nonspecific control stimulant. In some experiments, the tissue was incubated with phosphodiesterase inhibitors (1 mM theophylline, 0.1 mM IBMX in saline) for 5 min prior to stimulation. After a given time, we stopped the stimulation by shock-freezing the hearts on an aluminum block immersed in liquid nitrogen. cAMP was then extracted from the tissue (30 min at 707C) with ethanol containing 10 mM theophylline and 2.5 mM IBMX. The time course of the cAMP response was determined by varying the incubation times in steps, from 2 s to 12 min. Controls without peptide were performed for each incubation time. The peptide concentration was usually 10 05 M, but some experiments were also performed with lower doses. The experiments were usually carried out at room temperature, but some additional studies of the time course of the response were done at 07C. To prepare dose-response curves, the peptides were applied in concentrations from 10 08 M to 10 05M; the incubation time chosen for these experiments was 1.5 min. cAMP Radioimmunoassay The cAMP extracted from the tissue was measured by RIA. As trace we used a cAMP derivative that was iodinated with 125I by a chloramine T method as follows: About 1 nmol of 2 *O-monosuccinyladenosine 3 *:5 *cyclic monophosphate tyrosyl methyl ester in 10 ml 0.5 M phosphate buffer (pH 7) was combined with radioactive sodium iodide solution (1.5 mCi in 1-3 ml). Chloramine-T solution (5 ml of 2 mg/ml in 0.5M phosphate buffer) was added, and the solution was mixed for 30 s. Sodium metabisulfite (100 ml of 5 mg/ml in phosphate buffer) was added. After sitting for 5 min, the mixture was applied to a conditioned Sep-Pak cartridge (C18, Waters Associates) which was then washed with 20 ml of water, and the radiolabeled compound was eluted with 80% aqueous acetonitrile containing 0.05% TFA. A total of 5 ml was collected in 1 ml fractions. Usually the first fraction, containing the highest amount of radioactivity, was stored at 0207C and used for the RIA. The antiserum was applied in a final dilution of 1:21,000. The RIA buffer consisted of: 0.2M Tris, 0.06% dextran, 0.1% BSA, 10 mM EDTA, and 10 mM theophylline, pH 7.4. An aliquot (10– 50 ml) of the ethanol extracts was dried and redissolved in RIA buffer. The cAMP was acetylated with 5 ml acetic anhydride/ triethylamine (1:2 v/v) per 50 ml sample. The trace (10,000 cpm/ tube) and antiserum were added, and incubation was carried out overnight at 47C. Bound and free trace were separated by the addition of 1% BSA in RIA buffer, followed by precipitation of
the protein with ice cold ethanol for 1 h at 47C. After centrifugation, the supernatant was discarded, and the protein pellet was counted in a gamma counter. The hearts were blotted between paper tissues and weighed, and the cAMP values were related to those weights. The control levels of cAMP varied from one experiment to the next, ranging from 1 to 8 pmol/mg tissue. Therefore the data from any experiment were normalized with respect to the mean of the controls from that experiment. The t-test for independent samples was used to compare the cAMP levels of treated and untreated hearts; the p value was õ 0.01. Mechanical Recordings Whole Helix hearts were excised, a canula was inserted through the atrium into the ventricle, and this preparation was mounted in a setup similar to that described by Payza (19). The heart was perfused with saline which was delivered from one of 2 reservoirs (control and test) that were connected through a three-way valve to the canula. In contrast to previous experiments (19) the test agent was not delivered in a relatively small bolus. Rather, the hearts were perfused for up to 10 min with a solution containing peptide or forskolin, and this was done by merely turning the valve to the appropriate perfusion fluid. This procedure allowed us to compare the mechanical response and the change in cAMP level over the same time course. The reservoirs were mounted so that the height of the water column would be kept constant, precluding pressure artifacts. The mechanical activity of the myocardium was recorded with a force transducer which was attached to the aorta and connected to a polygraph (Grass model 79C). RESULTS
Effects of Cardioactive Agents on cAMP Levels Several cardioactive peptides and 5HT (all at 10 05 M) were tested for their effects on the cAMP levels in isolated ventricles. Forskolin (10 05 M), which activates adenylate cyclase directly, was also applied as a positive control (Fig. 1). SCPb increased cAMP levels markedly over control levels. In contrast to this robust response, that of 5HT was only modest, and no detectable effects were observed after stimulation with FMRFamide, the excitatory heptapeptide pQDPFLRFamide, or the inhibitory peptides pQDPFLRIamide and APGWamide. Since SCPb potently increases cAMP levels whereas FMRFamide is inactive, we tested the effects of three additional synthetic analogs: FPRMamide (the C-terminal tetrapeptide of SCPb), FPRFamide and FMRMamide. The latter two peptides are intermediate in structure between the C-terminals of the SCPs and FMRFamide. Of these analogs, only the two containing proline — FPRMamide and FPRFamide — significantly increased cAMP levels over controls (5.4- and 2-fold, respectively), and neither analog was nearly as active as SCPb. The very small increase produced by FMRMamide was not significantly different from controls (Fig. 2). Characterization of the SCPb Effect The time course of the increase in cAMP levels was distinctive. At room temperature, the response to SCPb was already maximal within 1-2 min, but this increase was followed by a fast decrease in cAMP levels which approached their control values asymptotically (Fig. 3). The control level of cAMP remained constant over the 12 minute duration of the experiment (not shown).
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FIG. 1. Effect of various cardioactive peptides, 5HT, and forskolin on myocardial cAMP. Each drug was applied for 1.5 min, except that forskolin was applied for 10 min. All drugs were used at a concentration of 10 05 M. Error bars Å SEM; * Å p õ 0.01. n Å 17,31 (SCPb, control); 6,19 (FMRFamide, control); 12,12 (pQDPFLRFamide, control); 8,8 (pQDPFLRIamide, control); 9,8 (APGWa, control); 7,15 (5HT, control); 10,8 (forskolin, control).
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FIG. 3. Time courses at 07C and room temperature for the stimulation of cAMP levels by SCPb (10 05 M). Error bars Å SEM. n Å 11 for room temperature; n Å 6 for 07C.
When the experiment was carried out at 07C, the time course was markedly different. The initial increase was slower, and the elevation of cAMP was lower; a maximal 7-fold elevation was measured after 6 min. The effect of the lowered temperature, in essence, was to keep the levels of cAMP elevated for a longer period. The effect of SCPb on cAMP levels was clearly related to dose (Fig. 4). Threshold was 10 08 M, and the maximal effect (an 8-15 fold increase over control levels) was produced by concentrations of 10 06 –10 05M. Application of phosphodiesterase inhibitors altered neither the threshold nor the relative shape of the time course at room temperature; but the maximal increase in cAMP was 5 to 6 times greater than without inhibitors (not shown). Mechanical Activity
FIG. 2. Structure-activity relations of the effect of SCPb on cAMP levels. FPRMa is the C-terminal tetrapeptide of SCPb; FMRMa and FPRFa are intermediates, each with 1 substitution. aÅamide. Doses were 10 05 M. Error bars Å SEM; * Å p õ 0.01. n Å 11,25 (FPRMa, control); 8,18 (FPRFa, control); 8,18 (FMRMa, control). Data for SCPb and FMRFamide are from Fig.1.
Ventricles were perfused with SCPb and forskolin, and the mechanical activity was recorded. In the past, drugs were injected as a bolus into the perfusion stream, so the effect lasted only for seconds. But because the increase in cAMP has a time course lasting minutes, we perfused the ventricles with test solutions for a longer period. Under these conditions, isolated Helix hearts respond to SCPb, as usual, with an increase of the systolic amplitude and a decrease of the diastolic tone; moreover the mechanical response persists for as long as SCPb is present (Fig. 5). Threshold for a mechanical activation by SCPb was 10 011 M, but individual differences occurred. At doses of peptide higher than 10 010 M the heartbeat became arrhythmic after 0.51 min of perfusion. In summary, the augmented mechanical cardioactivity caused by SCPb at room temperature is distinct — in both time course and threshold — from the phasic elevation of cAMP produced in isolated hearts by the same peptide.
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FIG. 4. Dose-response relationship of the SCPb effect on cAMP levels. Incubation time with peptide was 1.5 min at room temperature. n Å 15; error bars Å SEM.
Treatment of isolated hearts with forskolin (10 05M) produced an effect similar to that of SCPb; systolic amplitude increased and diastolic tone declined. But the cardioexcitation evoked by forskolin did differ from the SCPb effects in that it outlasted the actual perfusion time by more than 10 min. APGWamide inhibits the beat of isolated Helix ventricles at a threshold of 10 08 M to 2 1 10 08 M by decreasing both the amplitude and the diastolic tone (Fig. 6). A slight increase in dose to 4 1 10 08 M led to a complete cardiac arrest. After 5 min of washout, the hearts recovered and continued to beat spontaneously. DISCUSSION
The effects of cardioexcitatory peptides on the heartbeat of the snail are very similar; the mechanical responses are distinguishable only by differences in relative potency or in pattern of beat. In contrast — as this report shows — the intracellular effects of these same peptides are markedly different. In particular, although SCPb causes a robust increase in myocardial cAMP, other cardioexcitatory peptides lack this effect and, therefore, probably activate different second messenger pathways in the Helix myocardium. Among the cardioexcitatory peptides that have been identified in Helix, the SCPs are the most potent mechanical stimulators (21), and they also increase myocardial cAMP strongly. Since elevation of myocardial cAMP with forskolin is accompanied by an increase in mechanical activity similar to that caused by the peptide, the SCP-induced cardioexcitation is likely to be mediated by cAMP. The thresholds and the time courses of the mechanical stimulation and cAMP elevation are different, but the activation of adenylate cyclase through a G-protein linked receptor is only the first step in augmenting cardioactivity. The final response of the beating heart to SCPb may therefore be amplified, so that a dose
FIG. 5. Mechanical responses of Helix ventricles to SCPb and forskolin (auxotonic recording). The drugs were applied over 10 min. (period indicated by horizontal bar). Doses are concentrations in the perfusing saline.
higher than 10 010 M already leads to arrythmicity, whereas changes in cAMP cannot be detected biochemically at that dose. We speculate that, in the animal, the peptide is released in short bursts, so that arrhythmia is not established, even at higher local concentrations. The more sustained elevation of cAMP levels at lower temperatures might reflect a longer biological half life of the peptide agonist, or a decreased activity of phosphodiesterases. The effect of temperature on the amplitude of the cAMP increase is complex and cannot be addressed here. Although the effects of purified extracts on isolated Helix hearts provided the initial evidence for the existence of ‘‘small cardioactive peptides’’ (11,12), the actual peptides were first purified and sequenced from Aplysia californica (14,17), an opisthobranch mollusc. The physiology of the SCPs have been studied intensively in Aplysia: in the heart (13) and more recently in the accessory radula closer (ARC) muscle (4). Moreover, the excitatory effects on both tissues is mediated by cAMP. The cas-
FIG. 6. Mechanical response of Helix ventricles to APGWamide. Period of application of the peptide is indicated by bars. Dose is the concentration in the saline perfusing the heart.
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cade in the ARC muscle includes the activation of a cAMP-dependent kinase and the phosphorylation of the 750 kDa protein twitchin (22). Thus, the modulation of muscle activity by SCPs may be mediated by a cAMP pathway in all of the molluscan tissues that have been studied. In sharp contrast to the robust effect of the SCPs on cAMP levels, the effects of the FaRPs - both tetrapeptides and heptapeptides - were indistinguishable from the controls. This marked difference in the actions of the FaRPs and SCPs could not have been anticipated. Not only are the two types of peptides cardioexcitatory, they also share the C-terminal sequence (F-x1-R-x2-amide, where x1 and x2 are hydrophobic residues) and, on this basis, were even considered to have some familial relationship (17). More to the point, however, FMRFamide has been shown to increase cAMP levels in some other molluscan preparations. For example, the heart of the freshwater clam Lampsilis claibornensis is inhibited by FMRFamide (18), that of the estuarine clam Mercenaria mercenaria is excited (7), and in both cases the effect is accompanied by an increase of myocardial cAMP. And FMRFamide also activates adenylate cyclase in membrane preparations of squid optic lobes (3). Our finding that the synthetic tetrapeptides intermediate in structure between an SCP (-FPRMamide) and a FaRP (-FMRFamide) C-terminal, are also intermediate in their effects on cAMP levels suggests that the apparent structural similarities in the two families lack functional significance. The potency of the analogs in increasing myocardial cAMP is correlated with their activity on the beat of isolated snail ventricles: i.e., the proline containing analogs were 15 times (FPRMamide) and 5 times (FPRFamide) more potent cardioexcitors than FMRFamide (6). At present, the effects of FMRFamide and related peptides cannot be ascribed to any one signal transduction pathway within molluscs, because a variety of mechanisms - in addition to the increase in cAMP - have been observed in different molluscan preparations. For example, the excitatory effect of FMRFamide
on the heart of the marsh mussel Geukensia seems to involve a decrease of IP3 (1), but a small increase of IP3 seems to accompany the effect of FMRFamide on the tentacle retractor muscle of Helix aspersa (5). In Aplysia sensory neurons, FMRFamide was reported to activate a phospholipase A2 pathway with metabolites of arachidonic acid as second messengers (2). Finally, a FMRFamide gated sodium channel has recently been cloned from a Helix aspersa cDNA library of the nervous system, but expression of this channel was not observed in the heart (10). In Helix aspersa , the tetrapeptide and heptapeptide FaRPs can be distinguished by their bioactivity (21) and by their behavior in radioligand-receptor binding assays. Thus, the radioligand dYFnLRFamide was specifically displaced from Helix heart and brain membranes by the tetrapeptide FaRPs, but not by heptapeptides (19,20). The two distinct sets of molluscan FaRPs must therefore have different receptors, and their effects might well be mediated by different transduction pathways. The inhibitory effect of APGWamide on the beat of isolated ventricles indicates that a mechanism antagonistic to the excitatory peptides might be present. But a cardioregulatory role for APGWamide in Helix aspersa has not been established, and the actual peptide, though present in Helix pomatia (16), has yet to be identified in H. aspersa. In conclusion, the SCPs are the only peptides of the known cardioactive blend in Helix aspersa that increase the levels of cAMP; the second messengers of the other peptides remain unknown. ACKNOWLEDGEMENTS
This work was supported by NIH grant HL28440 to M.J. Greenberg and DFG grant Re1/1 to G. Reich. We are indebted to Dr. H.-Y.T.Yang for her generous gift of the cAMP antiserum. This is contribution number 316 from the Tallahassee, Sopchoppy & Gulf Coast Marine Biological Association.
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