Gen. Pharmac. Vol. 20, No. 3, pp. 381-384, 1989 0306-3623/89 $3.00 + 0.00 Printed in Great Britain. All rights reserved Copyright © 1989 Pergamon Press plc

EXCITATORY ACTION OF THE NATIVE NEUROPEPTIDE ANTHO-RFAMIDE ON MUSCLES IN THE PENNATULID

RENILLA KOLLIKERI

M. ANCTIL 1 and C. J. P. GRIMMELIKHULIZEN 2

~D6partement de sciences biologiques and Centre de recherche en sciences neuroiogiques, Universit6 de Montr6al, Montr6al H3C 3J7, Qu6bec, Canada and 2Center for Molecular Neurobiology, University

Hospital Eppendorf, PalviUion 22, D-2000 Hamburg 20, F.R.G.

(Received 7 June 1988)

Abstract--1. Antho-RFamide (pGlu-Gly-Arg-Phe-amide), a neuropeptide recently isolated from the sea pansy Renilla k611ikeri induced sustained (tonic) contractions in the rachis and peduncle of the colony, and in the individual autozooid polyps.

2. The threshold concentration for this effect was 5 nM in summer colonies and ! pM in autumn or winter colonies.

3. The peptide-induced tonic contractions were unaffected in sodium-free sea water. There was a 30% reduction of the contraction amplitude in sea water lacking calcium.

4. Peptides related to Antho-RFamide and other peptides were also examined for activity on rachidial muscles. Only peptides containing the carboxyterminal sequence Arg-Pbe-amide were active.

5. It is concluded that Antho-RFamide acts on Renilla muscles via a specific receptor and that it is a candidate for neurotransmitter or modulator in this pennatulid.

INTRODUCTION

Neuropeptides containing an Arg-Phe-amide-like carboxyterminus are abundant and widespread in coelenterates (Grimmelikhuijzen, 1985; Grimmelik- huijzen et al., 1986). Using immunocytochemistry with antisera to the sequence Arg-Phe-amide (RFa- mide), numerous immunoreactive neurons could be visualized in the pennatulid Renilla k611ikeri (Grim- melikhijzen and Anctil, in preparation). Using a radioimmunoassay for RFamide, a peptide was iso- lated from acetic acid extracts of Renilla and its sequence established as pGlu-Gly-Arg-Phe-amide (Grimmelikhuijzen and Groeger, 1987). Earlier, a neuropeptide with the identical sequence was isolated from the sea anemone Anthopleura elegantissima (Grimmelikhuijzen and Graft, 1986). Since these two phylogenetically widely separated anthozoan species produce the same neuropeptide, this substance is probably o f general occurrence in Anthozoa. For this reason we have called pGlu-Gly-Arg-Phe-amide "An tho -RFamide" .

The biological activity of the natural product Antho-RFamide has not been studied in pennatulids. Recently, Antho-RFamide was reported to have ex- citatory actions on muscles and conducting systems in the sea anemone Calliactis parasitica (McFarlane et al., 1987). Here we describe the effect of this peptide in the pennatulid Renilla kMlikeri.

MATERIALS AND METHODS

Renilla kMlikeri was obtained from Pacific Bio-Marine Laboratories, Venice, California. The colonies were main- tained in a steady current of artificial sea water (ASW, Wards) at 12-14°C. They were used in experiments within 3-4 wk of arrival.

Contractile activity of the rachis and peduncle was mon- itored as described in Anctil (1987). Briefly, pieces of rachis and peduncle were excised consistently from the same region of the colony, and pinned on Sylgard (Dow-Corning) in a perfusion chamber. Subsequently, the tension was recorded with a Grass FT-03C force displacement transducer, and the signals transmitted to a Grass 79D polygraph (Grass Instru- ments, Quincy, MA). For some experiments, insulated platinum electrodes, permanently fixed in the chamber, were used for electrical stimulation of the preparations. Some preparations consisted of one polyp (autozooid) left an- chored to a small piece of colony. The longitudinal con- tractile activity of the polyp was then recorded simulta- neously with that of the rachidial piece on separate polygraph channels.

The procedure for drug application was similar to that used by Anctil (1987). The pieces of colony or polyps were bathed in ASW at 16-20°C. All peptides were added directly to the bath, and all doses expressed as final molar concen- trations in the bath. The preparations were usually main- mined in contact with the active peptides until the maximum response amplitude was reached. After this, the prepara- tions were washed by draining and refilling the bath 3 times, and a different concentration of the same peptide or another peptide was added after the baseline tension was restored. In some preparations, the entire course of the contractile response, including relaxation, was allowed to unfold before evacuating the peptides. In the case of apparently inactive peptides, washing occurred 5 rain after adding them to the bath.

Peptides other than Antho-RFamide were tested on at least two rachidial pieces at a concentration of 10 ?tM. After these tests, Antho-RFamide was added to check the re- sponsiveness of the preparations. Peptides which showed some activity were subsequently tested on single prepara- tions to establish their dose-response curves. This was done with bath changes in between successive peptide concen- trations. Preparations were also repeatedly challenged with the same peptide concentration (1-10/~M) to eliminate the possibility of desensitization or facilitation. Response ampli- tudes were found to remain similar in such consecutive

381

382 M. ANCTIL and C. J. P. GRIMMELIKHULIZEN

STIM. A •

5 nM STIM.

A lOnM A t ~ n M

Fig. 1. Continuous recording of the contractile activity of a rachidial piece of Renilla kdllikeri (harvested in summer) which was subjected to electrical stimulation (A; 10 V, 10 ms duration, 2 Hz, 5 pulses) and exposed to increasing concentrations of Antho-RFamide (A). Antho-RFamide induces an increase in tonus. The preparation exhibits spontaneous rhythmic contractions which are not affected by Antho- RFamide. Note, however, that the amplitude of the eloctrically induced contractile response is markedly increased after exposure to 5 nM of peptide. Small arrow: increase in tonus accompanying a spontaneous rhythmic contraction while the preparation is exposed to the peptide. Vertical scale: 1.2 x 10 -6 N.

Horizontal scale: 3 min.

challenges. All these tests were conducted with summer colonies. From the EDso values, an "equiactive molar ratio" was calculated. This is the ratio of EDs0 of tested peptide versus Antho-RFamid¢. The EDs0 value for Antho- RFamide had been calculated from a dose-response curve obtained previously from another preparation for each set of experiments.

The substituted ASWs (sodium- and calcium-free) were prepared according to Wilkens (1972). Antho-RFamide was custom synthesized by Bachem (Buhendorf, Switzerland), and was pure as demonstrated by HPLC (Grimmelikhuijzen and Graft, 1986). pGlu-His-Gly-amide, pGlu-His-Pro- amide, pGlu-His-Gly, pGlu-His-Pro, Phe-Leu-Arg-Phe- amide, Phe-Met-Arg-L-Phe-amide, Phe-Met-Arg-D-Phe- amide, Phe-Gly-Gly-Phe, Tyr-Phe-Met-Arg-Phe-amide and Trp-Met-Asp-Phe-amide were from Sigma. All other pep- tides were custom synthesized by Bachem.

R E S U L T S

Antho-RFamide induced a tonic contraction in excised pieces of the rachis or peduncle, and in preparations of autozooid polyps, without affecting the ongoing rhythmic contractions. Typical record- ings of such contractions are shown in Fig. 1 for a summer colony. The response to Antho-RFamide appeared within 16 + 6 sec (n = 14) after bath appli- cation of the peptide. The total duration of the response was dependent on the peptide concen- tration. At low concentrations (5-10 nM), the initial tonus was restored 10 + 3 min (n = 10) after onset of response, and at higher concentrations (50 nM-1 ~tM) after 27 + 1 min (n = 10) [Fig. 1]. In the latter case, washing off the peptide while the tonic contraction was ongoing failed to quickly restore the tension to its resting level, and the duration of the decline in tension lasted several minutes whether or not the peptide was washed off.

For preparations made from summer animals (August-September) the average threshold concen- tration for inducing the increased tonus was 5 nM, although in 3 out of 9 preparations tested, a clear rise in tonus was already observed at 2nM. A dose-response curve for the peptide-induced con- traction shows that the half-maximal response was obtained at 100mM (Fig. 2). Preparations from Renilla colonies which were harvested during the fall

and early winter (October-January) showed re- sponses of similar shape and amplitude to Antho- RFamide as the preparations from summer animals (see Fig. 3). The sensitivity to Antho-RFamide, how- ever, was decreased 100-fold in fall-winter prepara- tions (Fig. 2).

In addition to the general increase in tone reported above, there was another rise in tonicity following each spontaneous rhythmic contraction in prepara- tions exposed to Antho-RFamide (small arrows: Figs 1 and 3). This effect was visible only at peptide concentrations of 10 nM and higher.

When contractions of rachis and peduncle were evoked by electrical stimulation, these contractions

lo0- D summer z~ win te r

75-

50-

. m X

0 - lo-; ~'~ lo-; 100~ 1o-~ lo-1

peptide concentration (M)

Fig. 2. Dose-response relationships of the tonic con- tractions elicited by Antho-RFamide in August (D) and Decemlxr (A) rachidial pieces of R. kdllikerL Peak ampli- tudes of the tonus change were measured, and the largest amplitude was used as reference (= 100% response). Each point represents the means (+ SEM) of the values recorded

in 6 separate experiments and preparations.

Bioactivity of Antho-RFamide in Renilla 383

Fig. 3. Effects of calcium substitution on the contractile responses of a single rachidial piece of R. kiillikeri (harvested in winter) to 5 PM Antho-RFamide. (A) Control response to Antho-RFamide (A), followed by washing off the peptide with fresh ASW (between large arrows). Note the increase in tonus following a rhythmic contraction (small arrow). (B) Continuous recording of the same preparation as in A, 1 h after substituting the ASW for calcium-free ASW. Note the disappearance of the spontaneous rhythmic contractions which gradually re-appeared after calcium-free ASW was replaced with normal ASW (between large arrows). Note that the response to Antho-RFamide is largely independent of external calcium. The small arrow in the lower trace again denotes a rise in tension following a spontaneous, rhythmic contraction after Antho-RFamide was added (open triangle). Vertical scale: 1.2 x 10e6N.

Horizontal scale: 3 mm

were also markedly enhanced in the presence of Antho-RFamide, even at concentrations as low as 5nM (Fig. 1)~

Application of Antho-RFamide to Renilla prepa- rations bathed in calcium-free sea water still induced a strong increase in tonus (Fig. 3B). This response invariably occurred in all 5 rachidial and 3 peduncle preparations thus tested. The peak amplitude of the peptide-induced tonic contraction in the absence of external calcium was reduced to 69 f 9% of controls (n = 8). In contrast to peptide-induced tonic con- tractions, the rhythmic contractions disappeared in

Table 1. Relative potency of Antho-RFamide and other peptides in the rachidial muscle preparation of Renilla kiillikeri

Peptide

pGlu-Gly-Arg-Phe-amide (Antho-RFamide) - pGIu-Gly-Arg-Phe-Gly-Tyr pGlu-His-Gly-amide pGlu-His-Pro-amide pGlu-His-Gly pGlu-His-Pro pGlu-Leu-Leu-Gly-GIy-Arg-Phe-amide Arg-Ala-amide Arg-Asn-amide Arg-Gly-amide Arg-Leu-amide Arg-Met-amide Arg-Phe-amide Arg-Pro-amide Arg-Ser-amide Arg-Trp-amide Arg-Val-amide Phe-Leu-Arg-Phe-amide Phe-Met-Arg-L-Phe-amide Phe-Met-Arg-n-Phe-amide Phe-Met-Arg-t_-Phe-OH Phe-Met-Lys-Phe-amide Phe-Gly-Gly-Phe Tyr-Phe-Met-Arg-Phe-amide Tyr-Gin-Gly-Arg-Phe-amide Tvr-Gin-Leu-Leu-Glv-Glv-Am-Phe-amide

Equiactive molar ratio*

1 - - -

- 40 - - - - - 50 - - - - 50 50

500

- 100 200 200

T&Met-Asp-Phe-arkda - - -

*For a definition, see Materials and Methods.

calcium-fee sea water (Fig. 3). Rachidial preparations bathed in sodium-free sea water were able to respond to Antho-RFamide in a manner similar to those bathed in calcium-free ASW (not shown). Although the rhythmic contractions vanished, the amplitude of the Antho-RFamide-induced contraction was within the range of control preparations (92 + IO%, n = 3).

Peptides other than Antho-RFamide also induced tonic contractions of the rachis, albeit with much less potency than the native peptide (Table 1). Only peptides containing the carboxyterminal sequence Arg-Phe-amide, such as pGlu-Leu-Leu-Gly-Gly-Arg- Phe-amide, Phe-Met-Arg-Phe-amide, Phe-Leu-Arg- Phe-amide and Arg-Phe-amide itself, showed this activity. The intact sequence Arg-Phe-amide ap- peared to be necessary for activity. Omission of the carboxyterminal amidation (such as in Phe-Met-Arg- Phe), or an exchange of the Phe for the other aromatic amino acid Tyr (Leu-Thr-Arg-Pro-Arg- Tyr-amide) or Trp (Arg-Tip-amide), all resulted in loss of activity (Table 1). The same was found when Arg was exchanged for the other positively charged amino acid Lys (Phe-Met-Lys-Phe-amide). Ex- changing the carboxyterminal L-Phe for D-Phe re- duced the activity of Phe-Met-Arg-Phe-amide by IO-fold.

DlSCIJSSlON

Antho-RFamide induced an increase in tonus in the muscular system of Renilla, and this response appears to be similar to that found in sea anemones (McFarlane et al., 1987). The action of Antho- RFamide was largely independent of external calcium (Fig. 3) or sodium, whereas rhythmic contractions were totally suppressed by calcium or sodium de- pletion in the sea water (Fig. 3). This suggests that Antho-RFamide acts directly on the muscle fibers and that intracellular pools of calcium ions must participate in the responses to Antho-RFamide (see

384 M. ANCTIL and C. J. P. GRIMMELIKHUUZEN

also Anctil, 1987). However, electrophysiological ex- periments on dissociated muscle cells will be neces- sary to dispel the possibility that some paracrine factor mediates the Antho-RFamide action. In view of the previous detection of large amounts of this peptide in Renilla (Grimmelikhuijzen and Groeger, 1987) and of the positive immunoreactivity of several sets of neurons to an antibody to Antho-RFamide in the same species (Grimmelikhuijzen and Anctil, in preparation), it is apparent that this neuropeptide is a serious neurotransmitter or modulator candidate in this pennatulid.

The molluscan neuropeptide FMRFamide and its fragment RFamide caused similar tonic contractions in Renilla as Antho-RFamide (Table 1 and Anctii, 1987). The sensitivity of Renilla tissue to its authentic neuropeptide Antho-RFamide, however, was 20-fold higher than that to either FMRFamide or RFamide. This could reflect a higher affinity of the Renilla muscle receptors for Antho-RFamide than for the other two peptides (see below). However, the stability of Antho-RFamide, which is protected at both the amino- and carboxytermini (pGlu and -amide) against unspecific amino- and carboxypeptidases, might also play a role. This stability will favour a high concentration of physiologically effective, intact peptide inside Renilla tissue.

The structure-activity data of Table 1 suggest that the action of Antho-RFamide is relatively specific. While the composition of the aminoterminus has only some minor effect on activity, any deletion or substi- tution in the carboxyterminal sequence Arg-Phe- amide leads to a complete or, in the case of the stereosubstitution of Phe, substantial loss. This sug- gests that receptor binding is involved in the action of Antho-RFamide. Although the details of their structure-activity data differ from ours, Price and Greenberg (1980) and Painter et al. (1982) have also documented the importance of the two carbox- yterminal residues of FMRFamide on activity in molluscan muscles.

The sensitivity of Renilla muscles for Antho- RFamide was markedly reduced in animals harvested during late autumn and early winter (Fig. 2). Similar

findings were also obtained for the sensitivity of Renilla muscles to FMRFamide (Anctil, 1987). The reasons for this variation are unclear.

Antho-RFamide does not play any apparent role in the spontaneous rhythmic contractions. However, the rise in tonicity associated with each rhythmic contraction in the presence of Antho-RFamide (small arrows, Figs 1 and 3) is an interesting phenomenon which deserves further study.

Acknowledgements--This research was supported by the Natural Sciences and Engineering Research Council of Canada (A6447), and the Deutsche For- schungsgemeinschaft (Gr 762/7).

REFERENCES

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