The Journal of Experimental Biology 202, 2595Ð2607 (1999) 2595 Printed in Great Britain © The Company of Biologists Limited 1999 JEB2314

CYCLIC AMP IS INVOLVED IN CARDIOREGULATION BY MULTIPLE ENCODED ON THE FMRFamide GENE

DEBBIE WILLOUGHBY*, MARK S. YEOMAN AND PAUL R. BENJAMIN Sussex Centre for Neuroscience, School of Biological Sciences, University of Sussex, Brighton BN1 9QG, UK *Present address: Department of Physiology, University of Cambridge, Downing Street, Cambridge, CB2 3EG, UK (eÐmail: [email protected])

Accepted 30 June 1999

Summary We have used a combination of biochemical and showed the inhibitory phase of the ‘isoleucine’ response, pharmacological techniques to investigate the role of the but characteristically lacked the delayed excitatory phase, cyclic nucleotides, 3′,5′-cyclic adenosine monophosphate were found to have high levels of membrane AC activity (cyclic AMP) and 3′,5′-cyclic guanosine monophosphate ജ10 pmoles min−1 mg−1 protein in controls. Application of (cyclic GMP), in mediating the cardioregulatory effects of the ‘isoleucine’ to membrane homogenate FMRFamide and other neuropeptides encoded on exon II preparation from these hearts failed to increase AC of the FMRFamide gene of Lymnaea stagnalis. The activity. The addition of FMRFamide produced significant ‘isoleucine’ peptides (EFLRIamide and pQFYRIamide) increases in the rate of cyclic AMP production in the heart produced complex biphasic effects on the frequency, force membrane preparations, which could account, at least in of contraction and tonus of the isolated heart of L. stagnalis, part, for the cardioexcitatory effects of this in the which were dependent on adenylate cyclase (AC) activity isolated whole heart. A membrane-permeable cyclic AMP of the heart tissue. At a control rate of cyclic AMP analogue (8-bromo-cyclic AMP) and an AC activator production of ഛ10 pmoles min−1 mg−1 protein, the (forskolin) were also cardioexcitatory. The peptide ‘isoleucine’ peptides produced a significant increase in AC SEEPLY had no effects on the beat properties of the activity in heart membrane preparations. This suggested isolated heart and did not alter AC activity. The activity of that the enhanced AC activity is responsible for the the membrane-bound (particulate) guanylate cyclase (GC) stimulatory effects of the ‘isoleucine’ peptides on frequency was not significantly affected by any of the peptides. and force of contraction of heart beat. This excitation sometimes followed an initial ‘inhibitory phase’ where the Key words: FMRFamide-related peptide, heart, 3′,5′-cyclic frequency of beat, force of contraction and tonus of the adenosine monophosphate, 3′,5′-cyclic guanosine monophosphate, heart were reduced by the ‘isoleucine’ peptides. Hearts that peptide signalling, multiple second messenger, Lymnaea stagnalis.

Introduction The majority of studies to date have shown that SEQPDVDDYLRDVVLQSEEPLY (known as ‘SEEPLY’). neuropeptides mediate their effects via the activation of a pQFLRIamide is not encoded by the FMRFamide gene (Kellett variety of different second messenger pathways, with each et al., 1994; Santama et al., 1995), but as this peptide is present signalling pathway playing an important role in producing the in both the motoneurones (Ehe cells) that provide the desired biological response (Lundberg, 1996). The second FMRFamidergic innervation of the heart and the heart tissue messenger-mediated effects resulting from the release of a itself (Worster et al., 1999), its effects were also examined in combination of peptides are likely to be highly complex and the present study. have been little studied. We chose to investigate this problem The previous paper showed a clear role for the calcium- further by examining the second messenger targets for a family mobilizing messenger, inositol-1,4,5-trisphosphate of neuropeptides, with diverse physiological actions, which are (Ins(1,4,5)P3), in mediating the excitatory effects of encoded on a single gene in Lymnaea stagnalis, the FMRFamide and FLRFamide in the heart (Willoughby et al., FMRFamide gene (Benjamin and Burke, 1994). The peptides, 1999). However, this messenger played no direct role in the encoded on exon II of the FMRFamide gene in L. stagnalis, mechanism of action of the two other classes of neuropeptides, were separated into three separate classes on the basis of their which are encoded on the same exon, and are known to be amino acid number and physiological effects: (1) FMRFamide present in the heart tissue. Two possible alternative second and FLRFamide, (2) EFLRIamide, pQFYRIamide and messenger targets were 3′,5′-cyclic adenosine monophosphate pQFLRIamide (referred to as the ‘isoleucine’ peptides) and (3) (cyclic AMP) and 3′,5′-cyclic guanosine monophosphate 2596 D. WILLOUGHBY, M. S. YEOMAN AND P. R. BENJAMIN

(cyclic GMP). In the present paper we examined the ability of radioimmunoassay (RIA) technique. Second, a series of the three classes of exon II peptide encoded on the pharmacological experiments were performed using the intact FMRFamide gene of L. stagnalis to regulate these two whole-heart preparation to compare the effects of the cyclic messenger systems. We attempted to relate changes in the nucleotides and peptide application. activity of these second messenger pathways to physiological actions of the neuropeptides on the heart. The cyclic nucleotides have been linked to the activity of numerous Materials and methods neuropeptides. Vasoactive intestinal peptide (VIP), Experimental animals , , small cardioactive peptide (SCP) and Lymnaea stagnalis (25Ð40 mm shell length) were supplied myomodulin have all been shown to upregulate the production by Blades biological (Kent, UK), or the Biology Department of cyclic AMP (Lloyd et al., 1985; Patterson et al., 1989; Weiss of the Vrije Universiteit (Amsterdam). All snails were et al., 1992). The peptides, and the locust maintained at 20 ¡C and subjected to a 12 h:12 h L:D cycle for F1 peptide have been shown to suppress the production of at least 14 days prior to experiments. Animals were fed ad cyclic AMP (Patterson et al., 1989; Baines et al., 1995). libitum on lettuce. Neuropeptides that induce increases in cyclic GMP production include , II, and atrial Synthetic peptides and pharmacological agents natriuretic peptide (ANP) (Reiser et al., 1984; Gilbert et al., FMRFamide and FLRFamide were purchased from Sigma 1986; Schmidt et al., 1993). These neuropeptides were shown (Dorset, UK). The peptides EFLRIamide, pQFYRIamide, to stimulate cyclic GMP levels indirectly, via the release of pQFLRIamide and SEQPDVDDYLRDVVLQSEEPLY calcium from intracellular stores (causing stimulation of nitric (‘SEEPLY’) were synthesized in our laboratory, and checked oxide synthase), or directly by interacting with the particulate for purity by HPLC. All other pharmacological agents were guanylate cyclase (GC) enzyme. In this study we examine the purchased from Sigma unless stated otherwise. effects of the peptides on the particulate GC enzyme only (not the soluble GC) using a heart membrane preparation. Saline and buffer composition Cyclic AMP has been shown to produce positive inotropic Hepes-buffered saline (HBS) (Benjamin and Winlow, 1981) −1 and chronotropic effects on mammalian, molluscan and contained (in mmol l ): NaCl (24), KCl (2), CaCl2 (4), MgCl2 hearts (Hartzell and Fischmeister, 1986; S.-Rózsa, 1980). In (2), NaH2PO4 (0.1), NaOH (35), glucose (1), Hepes (50), many molluscan hearts there is a correlation between the pH 7.9. Tris-DTT buffer (in mmol l−1): Tris-HCl (7.5), DTT −1 positive inotropic and chronotropic effects of 5-HT and SCPb (1), pH 7.0. Tris-DTT-EDTA buffer (in mmol l ): Tris-HCl with increased cyclic AMP levels (Higgins, 1974; (7.5), DTT (1), EDTA (1), pH 7.0. Assay solution (in Mandelbaum et al., 1979; Sawada et al., 1984; Lloyd et al., mmol l−1): Tris-acetate buffer (375) pH 7.6, IBMX (0.5), 1985; Reich et al., 1997). In the mammalian heart magnesium acetate (50), guanosine 5′-triphosphate (0.25). The acetylcholine stimulates cyclic GMP levels, or reduces cyclic IBMX acts as a phosphodiesterase inhibitor and prevents the AMP production, to produce negative inotropic effects (Tohse breakdown of both cyclic AMP and cyclic GMP. et al., 1995; Hartzell and Fischmeister, 1986). However, the effects of cyclic GMP in the invertebrate heart are poorly Cyclic nucleotide production in heart membrane preparation documented. In hearts from Locusta migratoria and Helix Hearts were rapidly dissected from snails, washed in normal pomatia high doses of cyclic GMP (10 mmol l−1) produced HBS and homogenized in 1 ml of ice-cold Tris-DTT-EDTA inconsistent effects on heart beat (S.-Rózsa, 1980). In L. buffer. 8Ð10 hearts were pooled to provide sufficient material stagnalis the effects of cyclic AMP and cyclic GMP on the for a single experiment. The homogenate was centrifuged at heart have not been examined. 13 000 revs min−1 (approximately 8000 g) for 5 min at 4 ¡C, Although nothing was known previously about the effects after which the supernatant was discarded and the pellet of the ‘isoleucine’ neuropeptides or SEEPLY on cyclic AMP resuspended in 1.5 ml of Tris-DTT-buffer. The homogenate or cyclic GMP levels there is evidence in molluscs that was centrifuged and resuspended twice more as described FMRFamide often acts via cyclic AMP. It has been shown that above. After each centrifugation the supernatant was removed. FMRFamide stimulates cyclic AMP levels and enhances The resulting membrane homogenate was stored for up to contractions in the bivalve heart and in gill tissue of Aplysia 4 months at −80 ¡C until required. At the time of use the californica (Higgins et al., 1978; Cawthorpe et al., 1985; Weiss membrane homogenate was resuspended in 1.5 ml of HBS, et al., 1985), and FMRFamide evokes a cyclic AMP-mediated from which 200 µl samples of heart homogenate were taken. decrease in K+ conductance in the E11 neurone of Helix All samples were kept on ice during the addition of 80 µl of aspersa (Colombaioni et al., 1985). the assay solution and 100 µl of peptide or HBS (control). 20 µl Two main approaches were used in the present investigation. of 50 mmol l−1 ATP or GTP was added to each sample, at First, the ability of FMRFamide, EFLRIamide, pQFYRIamide, 20 ¡C, to begin the reaction (ATP was used to stimulate cyclic pQFLRIamide and ‘SEEPLY’ to alter the rate of cyclic AMP AMP production, whilst GTP stimulated the production of and cyclic GMP production in a heart membrane preparation cyclic GMP). Samples of 50 µl were taken from each of the from L. stagnalis was analysed biochemically using a samples at 0, 5, 10, 20, 45, 60 or 120 s incubation time, and Multiple second messenger targets for neuropeptides 2597 pipetted into Eppendorf tubes in a hot-water bath, maintained their protein content would fall within the working region of at approximately 70 ¡C, to stop the reaction. Cyclic nucleotides the standard curve. (cyclic AMP and cyclic GMP) were extracted by keeping the 50 µl samples in the hot-water bath for a further 5 min, and then Whole-heart pharmacological experiments probe-sonicating for 3Ð4 s. The samples were finally Hearts were removed from the snails and mounted in an centrifuged for 5 min at 13 000 revs min−1 (approximately experimental perfusion chamber for isotonic recording as 8000 g) and their supernatants were diluted in 200 µl of described in Willoughby et al. (1999). The time of drug 0.5 mmol l−1 sodium acetate buffer. These samples were then solution perfusion varied between 30 s and 2 min, depending assayed to determine cyclic AMP or cyclic GMP production on the type of experiment. Normal HBS was perfused between using cyclic-nucleotide-specific RIAs. each application of drug for a minimum of 8 min to allow the heart to return to a steady beat rate. In addition, the outside of Cyclic nucleotide radioimmunoassay proceedure the heart was constantly perfused with HBS via a peristaltic 50 µl samples of cyclic nucleotide standards or sample pump. Contractions were permanently recorded on a brush supernatants were pipetted in duplicate and acetylated with chart recorder (Gould Instruments, Hainault, UK). A wide 5 µl (for cyclic AMP) or 2.5 µl (for cyclic GMP) of a mixture range of peptides and drugs were applied to the heart. All of triethylamine:acetic anhydride (2:1) to improve assay agents tested were dissolved in HBS and adjusted to pH 7.9. sensitivity. 50 µl of antibody and 50 µl of 125I-labeled cyclic nucleotide were then added to each standard or sample Statistics duplicate. The antibodies (provided by the Biochemistry Data are reported as mean ± S.E.M. Means were compared Department, University of Sussex, UK) were prepared in using the Student’s t-test. P<0.05 was considered significant rabbits against succinyl cyclic AMP/cyclic GMP-conjugated and N was the number of experiments performed in the RIAs, albumin. Cross-reactivity of the antisera with other cyclic or the number of hearts in the pharmacological experiments. nucleotides was less than 0.1 %. In addition, duplicate tubes were set up containing labeled cyclic nucleotide only (total count), and labeled cyclic nucleotide plus 0.5 mol l−1 sodium Results acetate buffer only (non-specific binding). The tubes were The ‘isoleucines’ and FMRFamide stimulate cyclic AMP incubated for a minimum of 18 h at 4 ¡C. Bound and free label production in the heart were then separated in each tube (with the exception of the The ‘isoleucine’ peptides (EFLRIamide, pQFYRIamide and total count duplicate) following the addition of 4 ml pQFLRIamide) and FMRFamide, applied separately, all polyethylene glycol/gamma globulin mix and a 20 min significantly increased the rate of cyclic AMP production in centrifugation at 30 000 revs min−1 (approx. 20 000 g) at 4 ¡C. homogenized heart membrane preparations from L. stagnalis After centrifugation the tubes were inverted to pour off the (Fig. 1). Concentrations of 1 µmol l−1 FMRFamide (N=21), supernatant and left to drain on absorbent paper for a 1 µmol l−1 EFLRIamide (N=21) and 1 µmol l−1 pQFYRIamide minimum of 1 min. The tops of the tubes were blotted dry by (N=15) produced similar maximum increases in the rate of gentle tapping of the inverted tubes against the tissue. The cyclic AMP production (AC activity) of 18.9±2.9, amount of bound cyclic nucleotide label in the resulting pellet, 19.7±2.5 and 19.4±2.9 pmoles cyclic AMP min−1 mg−1 protein, and the amount of radioactivity in the ‘total counts’, was respectively, at 5 s of peptide incubation. These values were determined using an RIA CALC program and an LKB- significantly greater than control levels of AC activity Pharmacia multi-gamma counter. Each sample was counted (P<0.001), which remained at approximately for 1 min and its cyclic nucleotide content was then 5Ð8 pmoles cyclic AMP min−1 mg−1 protein throughout the determined by comparison with a standard curve. 2 min incubation period. During peptide incubation AC activity steadily declined from the peak value at 5 s and slowly returned Protein assays towards control activity levels over the 120 s incubation period. The protein content (mg ml−1) of each homogenate The third ‘isoleucine’ , pQFLRIamide, also preparation used for the cyclic nucleotide radioimmunoassay stimulated AC activity significantly at 5 s of peptide incubation was determined using the Bio-Rad protein assay (Bradford (P<0.01, N=6) but was less potent than the other peptides. The method). Bovine plasma gamma globulin was used as peptide pQFLRIamide produced a maximum increase standard. 50 µl of each protein standard or sample of unknown in the rate of cyclic AMP production to protein content was pipetted into a cuvette with 450 µl distilled 13.8±2.4 pmoles cyclic AMP min−1 mg−1 protein (Fig. 1). water and then 100 µl of the dye-reagent. The contents were 10 µmol l−1 SEEPLY (N=11) did not significantly affect the then mixed by inversion. After approximately 15 min of rate of production of cyclic AMP in L. stagnalis heart incubation the absorbance at 595 nm was measured against the membrane homogenate (Fig. 1). The adenylate cyclase (AC) ‘blank’ sample (i.e. zero protein content) using a activator, forskolin (FSK) (1 µmol l−1) was used as a positive spectrophotometer. Unknown protein concentrations were control for cyclic AMP production and caused a fivefold determined using a standard curve. It was common for the increase in AC activity. At 5 s of forskolin incubation the samples to require dilution prior to the protein assay so that rate of cyclic AMP production was increased to 2598 D. WILLOUGHBY, M. S. YEOMAN AND P. R. BENJAMIN

25 25 ** ** 20 +FMRFamide 20 +EFLRIamide ** ** 15 15 * * 10 10

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20 50 Adenylate cyclase activity (pmoles cyclic AMP min 40 +FSK 15 +SEEPLY ** ** ** 30 ** ** 10 20 5 10

0 0 0 20 40 60 80 100 120 0 20 40 60 80 100 120 Time (s) Time (s)

Fig. 1. Increased rates of cyclic AMP production in heart membrane homogenate of L. stagnalis by exon II-encoded peptides. Adenylate cyclase activity (pmoles cyclic AMP min−1 mg−1 protein) was measured during incubation with 1 µmol l−1 FMRFamide (N=21), 1 µmol l−1 EFLRIamide (N=21), 1 µmol l−1 pQFYRIamide (N=15), 1 µmol l−1 pQFLRIamide (N=6), 10 µmol l−1 SEEPLY (N=11) and 1 µmol l−1 forskolin (FSK) (N=6). Values were recorded at 0, 5, 10, 20, 45 and 120 s of peptide incubation. The filled circles represent control − rates of enzyme activity. 5 mmol l 1 IBMX was used in all experiments to prevent cyclic nucleotide breakdown. Values are mean ± S.E.M. Asterisks indicate values significantly different from controls; *P<0.01, **P<0.001.

33.4±3.3 pmoles min−1 mg−1 protein (P<0.001, N=6) (Fig. 1). below 10 pmoles min−1 mg−1 protein. Other assays were A similar rate of cyclic AMP production was maintained performed on heart membrane with higher control AC throughout the remainder of the 2 min incubation period with activity (22.5±4.1 pmoles cyclic AMP min−1 mg−1 protein, 1 µmol l−1 forskolin. N=14). However, there was no increase in AC activity in The data analysed in Fig. 1, which show a clear stimulation response to the peptides in these batches of tissue of the rate of cyclic AMP production by FMRFamide and the homogenate, suggesting that the stimulatory effects of the ‘isoleucine’ peptides, were obtained from a heart membrane peptides were limited to tissue in which control AC activity homogenate with control rates of cyclic AMP production of levels were below 10 pmoles min−1 mg−1 protein. This is Multiple second messenger targets for neuropeptides 2599

1000 A 1000 B FMRFamide EFLRIamide 800 800

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-200 -200 0 15 304 5 0153045 Adenylate cyclase activity (pmoles min-1 mg-1 protein)

Fig. 2. Scatter plots showing the relationship between peptide-stimulated cyclic AMP production and control levels of adenylate cyclase activity (pmoles cyclic AMP min−1 mg−1 protein) in the heart. Each graph represents peptide-stimulated cyclic AMP production plotted as a percentage increase over controls (broken line) at 2 min of incubation. Cyclic AMP production during application of (A) 1 µmol l−1 FMRFamide, (B) 1 µmol l−1 EFLRIa, (C) 1 µmol l−1 pQFYRIamide and (D) 1 µmol l−1 pQFLRIamide to the heart homogenate.

supported by the data in Fig. 2, which shows scatter plots of measured. The threshold peptide concentration necessary to the percentage increase in total cyclic AMP production produce an increase in the rate of cyclic AMP production was evoked by a 2 min incubation with each of the four peptides, approximately 10 nmol l−1. Peptide concentrations of plotted against control AC activity. Analysis of the data in 100 µmol l−1 (the highest concentration tested) produced a this manner showed an apparent maximum rate of basal significant increase in the rate of cyclic AMP production for cyclic AMP production in the range of all three peptides when compared to controls (P<0.05, N=6). 10Ð15 pmoles min−1 mg−1 protein; above this level the The increases in cyclic AMP production rate seen in this set of peptides were unable to stimulate cyclic AMP production. experiments, particularly for EFLRIamide and pQFYRIamide, Importantly, in this heart membrane homogenate with high were less than those predicted from earlier experiments in AC activity, 1 µmol l−1 forskolin was still able to increase the which heart membrane homogenate was incubated for various rate of cyclic AMP production by almost threefold to times with 1 µmol l−1 concentrations of the peptides (Fig. 1). 62.3±7.4 pmoles min−1 mg−1 protein (P<0.01, N=3). Hence, This might be accounted for by the control rate of cyclic AMP although the tissue was apparently at ‘saturation’ for the rate production lying close to ‘saturation’ point. Dose–response of cyclic AMP production to be increased by the peptides, the data for SEEPLY (N=6) (Fig. 3A) showed no significant AC activity could still be directly stimulated by forskolin. changes in the rate of cyclic AMP production compared to Fig. 3 illustrates the ability of FMRFamide (N=6) (Fig. 3A), controls. EFLRIamide (N=6) and pQFYRIamide (N=6) (Fig. 3B), to In summary, it appears that the ‘isoleucines’ and stimulate the rate of cyclic AMP production in a concentration- FMRFamide might mediate some of their physiological effects dependent manner, over a concentration range known to via cyclic AMP. In the case of FMRFamide, this would be an produce physiological effects in the heart. Tissue membrane additional mechanism to the stimulation by this peptide of the homogenates were incubated with the peptide for 1 min and inositol phosphate signalling pathway (Willoughby et al., total cyclic AMP production over the 1 min period was 1999). 2600 D. WILLOUGHBY, M. S. YEOMAN AND P. R. BENJAMIN

180 A 70 B * FMRFamide EFLRIamide * 140 SEEPLY pQFYRIamide * 50 Fig. 3. Dose-dependent stimulation of cyclic 100 * AMP production in heart membrane homogenate * * 30 * by FMRFamide and the ‘isoleucine’ peptides. * DoseÐresponse curves for FMRFamide and 60 SEEPLY (A) and EFLRIamide and pQFYRIamide (B). All peptide incubations were 10 Cyclic AMP production

(% increase over control) 20 for 1 min, and 5 mmol l−1 IBMX was used in all experiments to prevent cyclic nucleotide breakdown. Values are mean ± S.E.M.(N=6). An -20 -10 asterisk indicates values significantly different 10-9 10-8 10-7 10-6 10-5 10-4 10-9 10-8 10-7 10-6 10-5 10-4 from control levels (broken line) (*P<0.05). [Peptide] (mol l-1)

The ‘isoleucines’ produce complex biphasic cardioregulatory effects Ai ‘Biphasic’ response Previous studies on the pharmacological actions of the E ‘isoleucine’ peptide EFLRIamide on isolated whole-heart I preparations indicated that it produced an initial slowing of heart beat followed by a longer lasting increase in beat frequency when perfused through the lumen of the L. EFLRIamide stagnalis heart (Santama et al., 1995). More detailed results Aii E on the actions of the ‘isoleucine’ peptides (EFLRIamide and I pQFYRIamide) were obtained in this study and are presented in Figs 4, 5. The effects of the ‘isoleucine’ peptides proved pQFYRIamide to be complex and variable, but could be summarized as dual action, causing both inhibitory and excitatory effects on heart beat. The biphasic responses to both EFLRIamide (N=10) and pQFYRIamide (N=5) (Fig. 4A) were comparable to those described by Santama and colleagues (1995). During the Bi ‘Inhibitory’ response inhibitory period the force of contraction was reduced and the heart beat slowed for a few seconds (Fig. 4Ai, I). The excitatory phase was more delayed and prolonged by EFLRIamide comparison (Fig. 4Ai, E). During this phase beat rate was increased and the tonus and force of contraction were also Bii increased in some preparations. In other hearts EFLRIamide produced purely inhibitory (N=13) (Fig. 4Bi) or purely pQFYRIamide Fig. 4. Pharmacological actions of the ‘isoleucine’ peptides in the isolated heart of L. stagnalis. (Ai) Biphasic response to perfusion of EFLRIamide (10 µmol l−1). There is an initial inhibitory phase (I), followed by a more prolonged excitation of heart beat (E). (Aii) Biphasic response to pQFYRIamide (10 µmol l−1) in the Ci ‘Excitatory’ response isolated heart. An initial weak inhibition is seen (I), followed by a prolonged, and more pronounced, excitation (E). (Bi) A potent µ −1 inhibitory response to EFLRIamide (10 mol l ) and (Bii) to EFLRIamide pQFYRIamide (10 µmol l−1). No excitatory effects were seen during perfusion of the ‘isoleucine’ peptides through both heart preparations. (Ci) Another isolated heart preparation produced a Cii purely excitatory response to 10 µmol l−1 EFLRIamide. (Cii) pQFYRIamide (10 µmol l−1) produced similar excitatory effects in pQFYRIamide this heart. Peptides were applied for the time indicated by the 0.5 g horizontal bars. The vertical scale bar calibrates the increases in underlying tonus of the hearts. 1 min Multiple second messenger targets for neuropeptides 2601 excitatory responses (N=8) (Fig. 4Ci), suggesting that the EFLRIamide (N=31) and pQFYRIamide (N=18) for all of the other type of response was absent or too weak to influence whole-heart preparations tested are shown in Fig. 5. heartbeat. Similar inhibitory (N=3) (Fig. 4Bii) or excitatory Statistical analysis of the data revealed that threshold responses (N=10) (Fig. 4Cii) in the heart were produced by concentrations of 100 nmol l−1 EFLRIamide and 1 µmol l−1 pQFYRIamide. The production of an inhibitory response by pQFYRIamide were required to cause a slowing of heart beat pQFYRIamide was less likely and was always weak (Fig. 5A). The maximal decrease in beat frequency was seen compared with that evoked by EFLRIamide (Fig. 4Bi). The during a 1 min application of 100 µmol l−1 EFLRIamide type of response seen for any particular heart was consistent (highest concentration tested) (P<0.01), or 10 µmol l−1 for repeated applications of the ‘isoleucine’ peptides. pQFYRIamide (P<0.05). Higher concentrations of Detailed doseÐresponse data for the inhibitory and excitatory pQFYRIamide (100 µmol l−1) produced purely excitatory components of the response seen during 1 min applications of responses. Increases in beat rate during the excitatory phase

‘INHIBITORY’ PHASE ‘EXCITATORY’ PHASE

[Peptide] (mol l-1) 10-9 10-8 10-7 10-6 10-5 10-4 100 D 0 ** A 80 -10 ** 60

-20 40 ** ** * 20 in beat frequency -30 ** Maximum % change 0 *** -40 ** 10-9 10-8 10-7 10-6 10-5 10-4

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20 -20

0

Fig. 5. DoseÐresponse curves for the in beat amplitude -30 inhibitory (AÐC) and excitatory (DÐF) effects Maximum % change of EFLRIamide and pQFYRIamide in the -20 10-9 10-8 10-7 10-6 10-5 10-4 isolated heart of L. stagnalis. (A) Maximum -40 ** percentage decrease in beat frequency, (B) maximum percentage decrease in beat -9 -8 -7 -6 -5 -4 100 F amplitude and (C) maximum decrease (mg) 10 10 10 10 10 10 ** 0 in tonus of the heart during the inhibitory phase seen during 1 min applications of the C 80 EFLRIamide (filled diamonds) and pQFYRIamide (filled circles). (D) Maximum -10 * 60 percentage increase in beat frequency, (E) * maximum percentage increase in beat *** 40 amplitude and (F) maximum increase (mg) in (mg) tonus during the excitatory phase in the same -20 20 experiments. All data values are mean ± ** S.E.M.(N=2Ð31 for EFLRIamide data, and Maximum change in tonus 2Ð18 for pQFYRIamide data). Asterisks 0 -9 -8 -7 -6 -5 -4 indicate values significantly greater than -30 10 10 10 10 10 10 controls; *P<0.05, **P<0.01, ***P<0.001. [Peptide] (mol l-1) 2602 D. WILLOUGHBY, M. S. YEOMAN AND P. R. BENJAMIN

(Fig. 5D) were seen at concentrations ജ100 nmol l−1 for both significantly different when compared to control values peptides, and were maximal during the application of (P<0.05 and P<0.01, respectively). 100 µmol l−1 EFLRIamide and pQFYRIamide (P<0.01), the highest concentrations tested. The effects of the peptides on Excitation of the heart by the ‘isoleucines’ is associated with beat amplitude were only seen during the inhibitory phase of an increased rate of cyclic AMP production the response (Fig. 5B). Maximal decreases in the beat It was hypothesized that the delayed excitatory actions of amplitude were produced by 10 µmol l−1 EFLRIamide or the ‘isoleucine’ peptides were related to the production of pQFYRIamide (P<0.01). Statistical analysis showed that cyclic AMP, a common mediator of excitatory effects in the beat amplitude was not significantly changed during the heart (Hartzell and Fischmeister, 1986; Lloyd et al., 1985). As excitatory phase of the ‘isoleucine’ response (Fig. 5E), predicted, hearts that produced a delayed excitatory response despite apparent increases in some preparations (in 5 out of to 10 µmol l−1 EFLRIamide (N=9) showed an increase in the 31 preparations for EFLRIamide; Fig. 4Ai, or in 4 out of 18 rate of cyclic AMP production in a membrane preparation of preparations for pQFYRIamide). During the inhibitory phase, the hearts. AC activity peaked at 5 s of EFLRIamide EFLRIamide (10 µmol l−1 maximal response), but not incubation, and remained higher than that seen in control tissue pQFYRIamide, significantly reduced the underlying tonus of throughout the 2 min incubation period (Fig. 6A). Hearts that the isolated heart muscle (P<0.01) (Fig. 5C). In contrast, both showed no delayed excitatory response to EFLRIamide peptides were capable of increasing the underlying tonus of perfusion (N=11) showed no increase in AC activity compared the heart during the delayed excitatory phase when to control (Fig. 6B). The rate of cyclic AMP production was EFLRIamide or pQFYRIamide were applied at determined, as described earlier, following examination of the concentrations >1 µmol l−1 (Fig. 5F). This ability of pharmacological response of the isolated hearts to 10 µmol l−1 pQFYRIamide and EFLRIamide to increase tonus was EFLRIamide. Hearts producing an excitatory or no excitatory

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EFLRIamide EFLRIamide

0.5 g 1 min

Fig. 6. Relationship between ‘isoleucine’-stimulated adenylate cyclase (AC) activity and cardioexcitation in L. stagnalis. (Ai) Stimulation of the AC activity in membrane homogenate from nine hearts showing excitatory responses to 10 µmol l−1 EFLRIamide. AC activity was analysed over a 2 min time course. (Aii) An example of the excitatory response (E) to a 10 µmol l−1 dose of EFLRIamide in one of the hearts pooled for cyclic AMP measurements. (Bi) Incubation with EFLRIamide for 2 min had no significant effect on AC activity in homogenate from 11 hearts that had previously shown no excitatory response to EFLRIamide. (Bii) An example of a purely inhibitory response (I) to 10 µmol l−1 EFLRIamide in one of the hearts pooled for cyclic AMP measurements illustrated in Bi. Peptides were applied for the time indicated by the horizontal bars. The vertical scale bar calibrates the change in underlying tonus of the heart muscle. Multiple second messenger targets for neuropeptides 2603 pharmacological response were divided into the two groups A FMRFamide and pooled. The hearts within each group were combined to provide sufficient tissue homogenate for a single RIA sample at 0, 5, 10, 20, 45 and 120 s of EFLRIamide incubation. The data from these two groups suggested that the potentiation of heart beat was dependent on control AC activity in the heart membrane homogenate. The membrane preparation from hearts showing excitatory responses to EFLRIamide exhibited, B EFLRIamide on average, a control rate of cyclic AMP production of approximately 5 pmoles min−1 mg−1 protein. In contrast, a membrane preparation of those hearts showing no excitatory response to EFLRIamide had a greater control rate of cyclic AMP production, around 15 pmoles min−1 mg−1 protein. This data is consistent with that shown in Fig. 2 and supports the C pQFYRIamide hypothesis that the increased rate of cyclic AMP production mediates the excitatory effects of the ‘isoleucine’ peptides. In contrast, the inhibitory effect of the isoleucines, shown for EFLRIamide in Fig. 6B, did not appear to be mediated by cyclic AMP. An interesting observation from these experiments was that hearts in which AC activity was higher D 8-Br-cAMP (15 pmoles min−1 mg−1 protein) had a higher resting beat rate (23±2 beats min−1) than those in which AC activity was lower (11±2 beats min−1 on average). This suggests that cyclic AMP might also have some role in the controlling the heart of L. stagnalis at rest.

8-bromo-cyclic AMP and forskolin produce excitatory effects E Forskolin in the heart To provide further evidence that cyclic AMP was responsible for the cardioexcitatory effects of the ‘isoleucine’ 0.5 g peptides and FMRFamide, the pharmacological effects of the 30 s peptides in the isolated heart were compared with those Fig. 7. Comparison of the effects of FMRFamide and the mediated by a membrane-permeable analogue of cyclic AMP, ‘isoleucine’ peptides with 8-bromo-cyclic AMP and forskolin in the 8-bromo-cyclic AMP (8-Br-cyclic AMP). As predicted 8-Br- isolated heart. (A) Excitatory response of the heart to cyclic AMP produced excitatory effects on the heart of L. 1 µmol l−1 FMRFamide application. (B) Biphasic response to stagnalis that were comparable to the delayed excitatory 10 µmol l−1 EFLRIamide with an initial inhibition of heart beat effects seen following application of the peptides (Fig. 7). followed by a more prolonged excitatory effect. (C) A predominantly Following a 10 s delay, 1 mmol l−1 8-Br-cyclic AMP mediated excitatory response of the isolated heart to 10 µmol l−1 pQFYRIamide increases in beat frequency and force of contraction of the application. (D) 1 mmol l−1 8-bromo-cyclic AMP(8-Br-cAMP) and − isolated heart (Fig. 7D). These effects were highly comparable (E) 100 µmol l 1 forskolin produced excitatory effects in isolated to the excitatory phase of the response of the heart seen heart preparations from L. stagnalis that were comparable to the following a 30 s perfusion of 10 µmol l−1 EFLRIamide excitatory effects seen during the application of the ‘isoleucine’ peptides, and some of the excitatory effects of FMRFamide. All (Fig. 7B) and 10 µmol l−1 pQFYRIamide (Fig. 7C) on the applications were for the time indicated by the horizontal bar. The heart. The cyclic AMP analogue also mimicked some of the vertical scale bar calibrates the increase in underlying tonus of the customary excitatory effects seen during a 30 s perfusion of the muscle. Data were obtained from different hearts. isolated heart with 1 µmol l−1 FMRFamide (Fig. 7A), except that it was slower to act than FMRFamide. An inhibitory response to 8-Br-cyclic AMP was never observed. Similarly, analogues, is consistent with studies in other molluscan hearts perfusion of the AC activator, forskolin, stimulated the heart (Higgins, 1974; Mandelbaum et al., 1979; Sawada et al., 1984). and produced increases in both the frequency and the amplitude of heart beat (Fig. 7E). The effects of 8-Br-cyclic RIA analysis of cyclic GMP levels AMP in the heart of L. stagnalis were concentration-dependent Activity of the particulate (membrane bound) GC enzyme (Fig. 8, N=11). The graphs illustrate the ability of 8-Br-cyclic was investigated using the heart membrane preparation. AMP to produce cardioexcitatory effects during 2 min SEEPLY, FMRFamide and the ‘isoleucine’ peptides had no applications of a range of concentrations (1 µmol l−1 to significant effect on the rate of cyclic GMP production in heart 1 mmol l−1). This cardioexcitation by cyclic AMP, or its membrane preparations from L. stagnalis. 2604 D. WILLOUGHBY, M. S. YEOMAN AND P. R. BENJAMIN

80 A Discussion (7) This study of peptidergic regulation of the heart in L. (9) *** stagnalis provides an important insight into the diversity of 60 *** peptide action in a well-characterized system. The results presented in this paper suggest that cyclic AMP is an important second messenger mediating the effects of the ‘isoleucine’ 40 peptides and FMRFamide in the heart. In the case of FMRFamide, this mechanism would be additional to the activation of inositol phosphate second messengers (7) 20 (Willoughby et al., 1999). The peptides EFLRIamide, * pQFYRIamide and FMRFamide all significantly increased the (2) rate of cyclic AMP production over control levels in responsive

Maximum % increase in beat frequency 0 tissue. The increases in AC activity were most prominent 10-7 10-6 10-5 10-4 10-3 within the first 5 s of peptide incubation. The third ‘isoleucine’ peptide, pQFLRIamide, which is not encoded by exon II of the FMRFamide gene but is present in the heart (Santama et al., 80 B 1995; Worster, 1999), also stimulated the rate of cyclic AMP production in heart membrane homogenate, but was less potent than the other peptides. 60 ** The stimulatory effects of EFLRIamide, pQFYRIamide, pQFLRIamide and FMRFamide on AC activity were only ** 40 apparent when control rates of cyclic AMP production were, on average, below 10 pmoles min−1 mg−1 protein. In batches of heart membrane homogenate with higher rates of cyclic AMP 20 production the peptides did not enhance further cyclic AMP production. In homogenate where the peptides had no effect on the rate of cyclic AMP production the ‘high’ basal rates of AC

Maximum % increase in beat amplitude 0 activity were comparable to the maximal rates of cyclic AMP 10-7 10-6 10-5 10-4 10-3 production produced by the peptides in tissue with ‘low’ basal AC activity. Forskolin significantly increased the rate of cyclic AMP production in heart membrane homogenates with both 80 C ‘low’ and ‘high’ levels of basal AC activity. The actions of forskolin in this tissue were presumably by direct stimulation of the AC enzyme. This suggested that ‘saturation’ of the 60 *** peptide-stimulated cyclic AMP production might occur *** upstream of the AC enzyme, for example at the G-protein or 40 at the peptide itself. Alternatively, it is possible that there are AC enzymes not associated with the ‘isoleucine’ receptor(s) that will still be stimulated by forskolin. In support 20 of the concept of saturation of second messenger production upstream from the AC enzyme are recent findings by Freedman et al. (1996), suggesting that high levels of intracellular cyclic Maximum increase in tonus (mg) 0 AMP are capable of uncoupling the β-receptor from its 10-7 10-6 10-5 10-4 10-3 stimulatory G-protein (Gs). The variability in control rates of [9-bromo-cyclic AMP] (mol l-1) cyclic AMP production between different batches of snails cannot be explained at present. Fig. 8. DoseÐresponse curves for the excitatory effects of 8-bromo- The pharmacological effects of 8-Br-cyclic AMP (a cyclic AMP in the isolated heart of L. stagnalis. (A) Maximum membrane-permeant cyclic AMP analogue) and forskolin (an increase in beat frequency plotted as percentage increase over AC activator) were comparable to the delayed excitatory controls, (B) maximum percentage increase in beat amplitude over effects of the ‘isoleucine’ peptides. The excitatory effects of 8- controls, and (C) maximum tonus change (mg) compared to the Br-cyclic AMP and forskolin were also comparable to the underlying tonus of the isolated heart in control conditions. Dose- dependent increases in all three parameters were seen during 2 min excitatory effects seen during perfusion of the isolated heart applications of the membrane-permeable cyclic AMP analogue. with FMRFamide. It seemed likely, therefore, that an increase Values are mean ± S.E.M.(N=2Ð9). Asterisks indicate values in cyclic AMP production might be responsible for the significantly greater than control; *P<0.05, **P<0.005, excitatory effects on heartbeat seen during application of both ***P<0.001. types of these peptides. The most striking similarity between Multiple second messenger targets for neuropeptides 2605 the cyclic AMP analogues and FMRFamide, EFLRIamide and receptor has been isolated in the squid (Chin et al., 1994). The pQFYRIamide was their ability to increase beat frequency. The identification of both a Gs- and a Gq-coupled FMRFamide maximal increase in beat frequency, of approximately 60Ð80 % receptor in the pond snail would account for the ability of the on average compared to the control beat rate, could similarly peptides to increase the production of both cyclic AMP and be achieved by applying cyclic AMP analogues or any of the Ins(1,4,5)P3 in the heart. Alternatively, there might be a single individual peptides. class of FMRFamide receptor in the heart of L. stagnalis that Detailed pharmacological effects of the ‘isoleucine’ is linked to two different G proteins, to give the subsequent peptides, EFLRIamide and pQFYRIamide, on the heart of L. activation of two separate signalling pathways. Wang et al. stagnalis were described. The ‘isoleucine’ peptides appeared (1995) suggested that both inhibitory and excitatory to have dual activity, producing complex biphasic responses in FMRFamide-related peptides (FaRPs) in the locust oviduct the heart consisting of both an inhibitory and an excitatory muscle might act via a single receptor linked to two different component. Similar data for the effects of EFLRIamide on the G proteins. However, definitive evidence for a single receptor heart of L. stagnalis have been reported previously by Santama in the intact muscle, rather than FaRPs actions via two et al. (1995), and in the related snail, Helix aspersa by Lesser pharmacologically similar receptor subtypes, was not and Greenberg (1993). The inhibitory effects of the presented. In vertebrates, β-adrenergic receptors have been ‘isoleucines’ dominated the early phase of the response to shown to couple with both Gs and Gi (Abramson et al., 1988; peptide application, reducing frequency and amplitude of heart Xiao et al., 1995), whilst the human thyrotropin receptor beat in particular. This was followed by a more prolonged couples to members of four G-protein families (Laugwitz et increase in beat frequency and tonus that appears to be al., 1996). Finally, it is also possible that the activation of one mediated by increased cyclic AMP production. Our RIA data second messenger pathway by a single transmitter substance show a peak increase in the rate of cyclic AMP production after stimulates the production of another second messenger. Studies 5 s of peptide incubation with heart membrane homogenate. in a range of neuronal cell types have identified three isoforms However, during such experiments the peptides have direct of AC that are stimulated by elevated levels of intracellular access to membrane-associated receptors. It is likely that the calcium (Cooper et al., 1995), such as the increase that might peak increase in cyclic AMP production occurs later, when the be seen following Ins(1,4,5)P3 production. Alternatively, peptides are perfused through the intact heart. Indeed, this studies by Prier et al. (1994) on heart of Manduca sexta showed would be more consistent with our pharmacological data, that the effects of the cardioacceleratory peptides (CAP2s), where the peptides often take longer to produce a clear which act via the production of Ins(1,4,5)P3, may be cardioexcitatory response. upregulated via an octopamine-sensitive cyclic AMP- In several of the hearts only inhibitory or excitatory effects dependent mechanism. The authors suggested a multiplicative were observed. Our data show a clear relationship between an interaction of the two pathways at the site of intracellular increased rate of cyclic AMP production and the excitatory calcium release. phase in hearts with relatively ‘low’ control AC activity levels. In L. stagnalis, we believe that the activation of two different The rate of cyclic AMP production in membrane homogenate signalling pathways, probably acting in parallel, can account from hearts showing no excitatory response to the ‘isoleucine’ for FMRFamide-induced cardioexcitation. Noradrenaline peptides was not stimulated further by the application of similarly activates these two signalling pathways in the peptides. vertebrate heart to mediate cardioexcitation (Irisawa et al., At present the inhibitory phase of the ‘isoleucine’ response 1993; Hartzell and Fischmeister, 1986; Difrancesco and cannot be accounted for by any of the second messengers Tortora, 1991). analysed in this or the previous paper, and might be due to The rate of production of cyclic GMP via the particulate GC direct effects of the peptides at an ion channel. Green et al. enzyme in the L. stagnalis heart was not significantly effected (1994) have identified two types of amiloride-sensitive Na+ by any of the peptides encoded by exon II of the FMRFamide channel in the C2 neuron of Helix that are directly gated by gene of L. stagnalis. We have not yet investigated whether the FMRFamide. A homologue of this channel has recently been peptides modulate activity of a soluble form of GC linked to cloned in our laboratory in L. stagnalis. Although activity of the production of nitric oxide, or indeed whether such a form the FMRFamide-gated Na+ channel could not account for the of GC enzyme is present in the heart of L. stagnalis. Perfusion inhibitory effects of the isoleucines, it is possible that a novel of isolated whole heart preparations with a membrane- isoleucine-gated channel might also exist, which could account permeable cyclic GMP analogue (8-bromo-cyclic GMP) (N=6, for the inhibitory response in the heart. data not shown) produced a relaxation of the heart. In The potential for FMRFamide to stimulate the production of particular, 8-bromo-cyclic GMP mediated a reduction in the cyclic AMP and inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) underlying tonus of the muscle. It is possible that the inhibitory (Willoughby et al., 1999) suggested that there might be two effects of the ‘isoleucine’ peptides might be mediated by different receptors for FMRFamide linked to second stimulation of the soluble GC and cyclic GMP production, but messenger pathways in the heart of L. stagnalis. As yet the this has not yet been investigated. FMRFamide receptor(s) of the heart of L. stagnalis have not In summary, the results obtained from this study provided been characterized, although a Gs-coupled FMRFamide sufficient evidence to indicate activation of at least two second 2606 D. WILLOUGHBY, M. S. YEOMAN AND P. R. BENJAMIN messenger pathways in the molluscan heart by peptides Neurotensin and its analogues − correlation of specific binding with encoded on exon II of the FMRFamide gene of L. stagnalis. with stimulation of cyclic GMP formation in neuroblastoma clone The excitatory effects of the ‘isoleucines’ are likely to be N1E-115. Biochem. Pharmacol. 35, 391Ð397. mediated by enhanced cyclic AMP production, while Green, K. A., Falconer, W. P. and Cottrell, G. A. (1994). The FMRFamide produces more potent excitatory effects through neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFamide) directly gates two ion channels in an identified Helix neurone. Pflugers. Arch. the apparent activation of both cyclic AMP- and InsP3- 428, 232Ð240. producing pathways. The modulatory peptide, SEEPLY, has Hartzell, H. C. and Fischmeister, R. (1986). Opposite effects of no significant direct effect on cyclic nucleotide and inositol cyclic GMP and cyclic AMP on Ca2+ current in single heart cells. phosphate production but could modify the production of these Nature 323, 273Ð275. second messengers by other exon II peptides. Other work in Higgins, W. J. (1974). Intracellular actions of 5-hydroxytryptamine our laboratory has suggested that the exon II-encoded peptides on the bivalve myocardium. I. Adenylate and guanylate cyclase. J. of the FMRFamide gene may be coreleased from the heart. The Exp. Zool. 190, 99Ð110. functional consequences of the release of such a ‘cocktail’ of Higgins, W. J., Price, D. A. and Greenberg, M. J. 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