the salmfamides: a new family of neuropeptides isolated from an echinoderm

The SALMFAmides: A New Family of Neuropeptides Isolated from an EchinodermAuthor(s): Maurice R. Elphick, David A. Price, Terry D. Lee and Michael C. ThorndykeSource: Proceedings: Biological Sciences, Vol. 243, No. 1307 (Feb. 22, 1991), pp. 121-127Published by: The Royal SocietyStable URL: http://www.jstor.org/stable/76708 .

Accessed: 07/05/2014 19:41

Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at .http://www.jstor.org/page/info/about/policies/terms.jsp

.JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range ofcontent in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new formsof scholarship. For more information about JSTOR, please contact [email protected].

.

The Royal Society is collaborating with JSTOR to digitize, preserve and extend access to Proceedings:Biological Sciences.

http://www.jstor.org

This content downloaded from 169.229.32.136 on Wed, 7 May 2014 19:41:51 PMAll use subject to JSTOR Terms and Conditions

http://www.jstor.org/action/showPublisher?publisherCode=rsl

http://www.jstor.org/stable/76708?origin=JSTOR-pdf

http://www.jstor.org/page/info/about/policies/terms.jsp


The SALMFamides: a new family of neuropeptides isolated from an echinoderm

MAURICE R. ELPHICK1'2, DAVID A. PRICE1, TERRY D. LEE3 AND

MICHAEL C. THORNDYKE2t 1 The Whitney Laboratory, University of Florida, 9505 Ocean Shore Boulevard, St. Augustine, Florida 32086, U.S.A. 2 Biology Department, Wolfson Building, Royal Holloway and Bedford New College, University of London, Egham, Surrey TW20 OEX, U.K. 3 Division of Immunology, Beckman Research Institute of the City of Hope, 1450 E. Duarte Road, Duarte, California 91010, U.S.A.

SUMMARY

We have isolated two novel related neuropeptides from the radial nerve cords of the starfishes Asterias rubens and Asteriasforbesi. One is an octapeptide with the amino acid sequence Gly-Phe-Asn-Ser-Ala-Leu- Met-Phe-NH2 and the other is a dodecapeptide with the amino acid sequence Ser-Gly-Pro-Tyr-Ser-Phe- Asn-Ser-Gly-Leu-Thr-Phe-NH2. The peptides were purified using high performance liquid chromato-

graphy (HPLC) and a radioimmunoassay for the molluscan FMRFamide-related neuropeptide, pQDPFLRFamide. Both peptides share minimal sequence identity with members of the family of FMRFamide-like peptides so we have designated them as founder members of a new family, the SALMFamides. We refer to the octapeptide as SALMFamide 1 (SI) and the dodecapeptide as SALMFamide 2 (S2). S1 and S2 are the first neuropeptides identified in species belonging to the phylum Echinodermata.

1. INTRODUCTION peptides, isolated only from protostomian species (molluscs, arthropods, annelids and nematodes), which

Peptides are probably the most ancient and ubiquitous share a C-terminal tetrapeptide sequence FXRFamide intercellular signalling agents. Peptidergic neurons are (X is Met, Leu or Isoleu) with FMRFamide. The

present in the nervous systems of all animals from FXRFamides are considered to constitute a family of coelenterates to mammals and are involved in the homologous neuropeptides unique to the protostomes regulation of a variety of physiological processes (Price & Greenberg 1989). (Thorndyke & Goldsworthy 1988; Holmgren 1989). Other FMRFamide-like peptides have been isolated The simple nervous systems of some invertebrate from both protostomian and deuterostomian phyla species provide model preparations for the study of and share the C-terminal RFamide, MRFamide or

many aspects of neuropeptide biology (O'Shea & LRFamide sequence with the FXRFamides, but lack a Schaffer 1985). One of the best characterized in- phenylalanine residue at the fourth amino acid position vertebrate neuropeptides is FMRFamide (Phe-Met- from the C-terminal amide. The widespread phylo- Arg-Phe-NH2) which was first detected in extracts of genetic distribution and limited sequence identity of molluscan ganglia through its cardioexcitatory activity these peptides suggests that they are not closely related

(Price & Greenberg 1977). FMRFamide is now and do not represent an interphyletic family of

recognized as a transmitter or modulator in identified homologues like the FXRFamides. They do, however neurons of the central and peripheral nervous systems include subsets of molecules that probably are hom- of molluscs (Brussaard et al. 1989; Cottrell 1989; Man- ologous because these share additional structural

Son-Hing et al. 1989). identity and are restricted to a single phylum. For Since FMRFamide was identified, antisera have been example, antho-RFamide and the pol-RFamides in

raised to it and used extensively for immunochemical coelenterates; A18Fa, F8Fa and LPLRFamide in studies. Such studies have revealed that FMRFamide- vertebrates and the leucosulfakinins in insects (Price & like immunoreactivity is present in the nervous systems Greenberg 1989). Relations between the FMRFamide- of species from most animal phyla (Boer et al. 1980; like peptides will be clarified when the structures of the

Grimmelikhuijzen et al. 1982). Purification and genes that encode them are compared and additional

sequencing of the immunoreactive substances has peptides are identified in phyla other than those revealed a structurally and phylogenetically diverse set already investigated. of FMRFamide-like peptides. These include a subset of One neglected phylum is the Echinodermata (e.g.

starfish, sea urchins, brittlestars and sea cucumbers) t To whom correspondence should be sent. and neuropeptides have yet to be identified in these

Proc. R. Soc. Lond. B (1991) 243, 121-127 Printed in Great Britain.

121



122 M. R. Elphick and others Echinoderm neuropeptides

animals. The echinoderms are of particular phylogenetic interest because they, along with the proto- chordates (e.g. sea squirts), are the only extant non- vertebrate deuterostomes. We have recently shown, using antisera to FMRFamide, that there is an abundance of neurons in the radial nerve cords of the starfish Asterias rubens which exhibit FMRFamide-like

immunoreactivity (Elphick et al. 1989 a). These neurons

appear to innervate the starfish locomotory organs (tube feet) and we speculate that the immunoreactive

peptides may be involved in the control of locomotion in these animals. We have now isolated and sequenced the peptides responsible for this immunoreactivity in both Asterias rubens and Asteriasforbesi and report their structures here. Some of these results have been

presented previously in abstract form (Elphick et al. 1989 b).

2. METHODS

(a) Peptide extraction

Two species of starfish were studied: Asterias rubens collected at the University of London Marine Bio-

logical Station, Millport, Isle of Cumbrae, Scotland

(a) 300-

Iq _ 200-

100- " -

C , J

(b)

280nm 210nm

and Asterias forbesi collected at the Marine Biological Laboratory, Woods Hole, Massachusetts. Radial nerve cords were dissected from the animals using the method described by Chaet (1964) and peptides extracted. Two different extraction methods were used for each starfish species. Nerve cords (5 g wet weight) dissected from 22 specimens of A. rubens were boiled in 50 ml of acetic acid (3 % by volume) for 10 min, homogenized and centrifuged. The supernatant was decanted and frozen (-30 C). Nerve cords (4 g wet weight) dissected from 80 specimens of A.forbesi were extracted in 16 ml of acetone overnight at 4 ?C. After removal of the acetone by rotary evaporation, the aqueous fraction was centrifuged and the supernatant frozen (-30 ?C).

(b) Peptide purification

The crude extracts were assayed for FMRFamide- like peptides using a radioimmunoassay (RIA). The RIA

protocol used has been described extensively by Price

(1982) and Price et al. (1985). Four antisera were used to test the extracts: L135 (anti-FMRFamide, from G. J. Dockray, Liverpool University) and S253 (anti- YGGFMRFamide, from D. A. Price) using 125I

5 10 15 20 25 30 / time/min

D E

(c) E

0 012 - 0.3 -

Q

0.4:

0-004- 0-- 2.3-

FLRF amde'equivat ents6 280nm ^ * 200

in RI A p moLesl 30

0 5 10 15 20 25 30 0 5 10 15 20 25 30 c-

time/min time./min

Figure 1. Purification of the SALMFamides from an acetic acid extract of radial nerves from Asterias rubens by RIA

and HPLC. (a) HPLC step 2. Gradient is 16-40% acetonitrile in 0.I % TFA over 30 min with fractions collected every 30 s. Two main immunoreactive peaks (D and E) and two smaller peaks (B and C) were detected. Peaks D and E (fractions 26-30) were pooled, separated in HPLC step 3 and further purified. (b) HPLC step 6 on peak D. HPLC conditions as in (a). The major immunoreactive fraction (28) contains about 60 pmol of peptide based on a FLRFamide standard curve and corresponds to a pure optical density (OD) peak that absorbs at both 210 and 280 nm. The identity of peak D was established by sequencing and FABMS (figures 3 a, b). (c) HPLC step 4 on peak E. HPLC conditions as in (a). Three other immunoreactive peaks were (A-C) detected in addition to peak E. The elution times of B and C correspond to those of peaks B and C in step 2. The major immunoreactive fraction (19) comprising peak B contains about 100 pmol of peptide based on a FLRFamide standard curve and corresponds to a pure OD peak that absorbs at 210 nm. Peak B was identified as the pentapeptide SALMFamide, by sequencing and FABMS (Figures 3c, d). The filled arrow indicates the elution time of synthetic SALMFamide and the open arrow indicates the elution time of oxidized synthetic SALMFamide.

Proc. R. Soc. Lond. B (1991)

n



Echinoderm neuropeptides M. R. Elphick and others 123

labelled YGGFMRFamide as the trace; Q2 (anti- pQDPFLRFamide, from D. A. Price) and L197 (anti- LPLRFamide, from G. J. Dockray) using 125I labelled

pQYPFLRFamide as the trace. The Q2 antiserum detected more immunoreactivity in the extracts than the other antisera so this antiserum was used in all further work.

The extracts were loaded onto a Brownlee RP300 C8 column (4.6 x 30 mm) using a Gilson HPLC system and eluted with a linear gradient of 0-60 % acetonitrile in 30 mM sodium phosphate (pH 7.0) over 30 min at a flow rate of 2.0 ml min-1. One minute fractions were collected and assayed using the RIA. Immunoreactivity in extracts from both starfish species was found to elute at between 12 and 14 min and was quantified as

pmoles of peptide based on a FLRFamide standard curve with the Q2 antiserum (see Bewick et al. 1990). For each species the immunoreactive fractions were

pooled, reapplied to the HPLC and further fractionated on a Brownlee RP300 C8 column (2.1 x 220 mm). A linear gradient of 16-40% acetonitrile in 0.1% trifluoroacetic acid (TFA) over 30 min at a flow rate of 0.5 ml min"1 was used and half minute fractions collected and assayed. This second HPLC step resolved

several immunoreactive peaks. Each of these peaks were purified to homogeneity separately by repeated fractionation on the C8 column (2.1 x 220 mm). The number of HPLC steps required and gradients used

varied, but in general it was found that alternating between the acetonitrile/TFA (16-40%) and aceto-

nitrile/phosphate (0-60%) gradients was the most effective method for purifying the peptides. Immuno- reactive peaks that corresponded to single optical density peaks on the HPLC were identified by automated Edman degradation sequencing and Fast Atom Bom- bardment Mass Spectrometry.

3. RESULTS

HPLC of the nerve extracts separated two main immunoreactive peaks (D and E in figures 1 and 2) as well as a variable number of smaller peaks (A, B and C in figure 1; C in figure 2). These smaller peaks re-

appeared de novo during later purification steps on peak E so must be modified forms of E. We first obtained a

pure peak of B and identified this as Ser-Ala-Leu-Met- Phe-NH2 (SALMFamide) by sequencing and Fast Atom Bombardment Mass Spectrometry (FABMS)

E

D

7- I

5 10 15 / time/min

L 20 25 30

E

(b)

280nm 210nm

0.0100- 0-28-

0-0075- 021-

0,0050- 014-

i 0-0025- 0-07-

0- 0/

D

5 1C

FLRF amide equivatents 75 in RIA i pmotes) I 210nm

280nm

15 20 25 30

time/min

(C) 0220-

E i 0-160

Q :

' 0-055:

1 :

E

150 F L RF amide equivalents ^ 1100 in R IA (p moes )

~. 50

5 10 15

time/mnin

20 25 30

Figure 2. Purification of the SALMFamides from an acetone extract of radial nerves from Asteriasforbesi by RIA and HPLC. (a) HPLC step 2. Gradient is 12-48% acetonitrile in 30 mM sodium phosphate over 30 min with fractions collected every 30 s. Two immunoreactive peaks detected (D and E); peak D (fraction 37) and peak E (fractions 42 and 43) were further purified separately. (b) HPLC step 4 on peak D. Isocratic conditions, 20% acetonitrile in 0.1 % TFA over 30 min with fractions collected every 30 s. The major immunoreactive fractions (22 and 23) contain about 30 and 40 pmol of peptide respectively based on a FLRFamide standard curve and correspond to a pure OD peak that absorbs at both 210 nm and 280 nm. The identity of peak D was established by sequencing and FABMS (figures 4a, b). (C) HPLC step 4 on peak E. HPLC conditions as in (b). Immunoreactive peak (C) detected in fractions 16 and 17 as well as peak E (fractions 26 and 27). Peak C was also generated in the next HPLC step (5) on peak E and identified as the oxidized form of peak E (Figures 4e,f). The immunoreactive fractions comprising peak E (26 and 27) each contain about 135 pmol of peptide based on a FLRFamide standard curve and correspond to a pure OD peak that absorbs at 210 nm. The identity of peak E was established by sequencing and FABMS (figure 4c, d) after further

purification.


(a)

X 200- ;;cr,

150- "

100

500

Ft 0

D

r -- .. , I , , I w s s 8 *

c




-e-- S -*-- G

-.-- P

y --- N

Fr N

3 4 5 6 7 8

cycle

(b) 10-

Z 8- . 7

z _= 6- CO

?4 . r

4

.- _ 1275

1000 1200 1400

nm/

(d)

-e--O- S ---A

---- *- L

-0 M

-U- F

a)

Co C C

22

F

5 6 7 8 550 600

ml/z

Figure 3. Identification of the SALMFamides in Asterias rubens by automated Edman degradation and fast atom bombardment mass spectrometry. (a) and (c) show the PTH amino acids from Edman degradation on peaks D and B respectively. In (a), a very small amount of peptide was available for sequencing; only the most abundant amino acid in each cycle is illustrated because all the other amino acids were either not detected or were present at background levels. (b) and (d) show the mass of the predominant molecular ion (M + H)+ in fast atom bombardment mass spectra of peaks D and B respectively. The sequence SGPYSFNS is not consistent with the mass 1275 for peak D but this mass is consistent with the sequence established for peak D from (a). A. forbesi, Ser-Gly-Pro-Tyr-Ser-Phe- Asn-Ser-Gly-Leu-Thr-Phe-NH2 (figure 4a). The sequence SALMF and mass 567.3 identifies peak B as Ser-Ala-Leu- Met-Phe-NH2.

(figure 3c, d). Synthetic SALMFamide and peak B co- elute from the HPLC column, whereas oxidized SALMF- amide elutes earlier than the reduced form and coincident with peak A (figure 1 c).

Peak E was first purified from an acetone extract of radial nerves from A.forbesi and identified as Gly-Phe- Asn-Ser-Ala-Leu-Met-Phe-NH2 (GFNSALMFamide) by sequencing and FABMS while peak C was shown to be the oxidized form of this peptide (figure 4c-f). Peak B (SALMFamide) was probably generated from GFNSALMFamide artefactually under the acid conditions employed in our initial extraction procedures and in some of the purification steps. More recently we have purified GFNSALMFamide from acetone extracts of A. rubens using an antiserum raised to KYSALMFamide (M. R. Elphick, J. R. Reeve & M. C. Thorndyke, unpublished observations).

Peak D from both starfish species exhibited UV absorbance at 280 nm as well as 210 nm (figures b and 2 b), so we expected that the peptide would contain a tyrosine or tryptophan residue. Sequencing and FABMS identified the peak in each species as Ser-

Gly-Pro-Tyr-Ser-Phe-Asn-Ser-Gly-Leu-Thr-Phe-NH2 (SGPYSFNSGLTFamide) (figures 3a, b and 4a, b).

It is, perhaps, worth pointing out that the sequencing was carried out at three independent laboratories

(Gainesville, Florida; City of Hope, Duarte, California and Egham). All three produced matching sequences

and the predicted structures were in all cases confirmed

by FABMS.

4. DISCUSSION

We have isolated two novel neuropeptides, GFNSALMFamide and SGPYSFNSGLTFamide, from extracts of radial nerve cords from two species of starfish, Asterias rubens and Asterias forbesi, through the use of a radioimmunoassay designed to detect FMRFamide-like peptides. Both peptides share the C- terminal Phe-amide with all FMRFamide-like peptides and the pre-penultimate leucine residue with some of them. However, a penultimate arginine residue is common to all members of the set of FMRFamide-like

peptides and its absence from this position in the molecules isolated from Asterias suggests that these are unrelated to this set of neuropeptides. It also confirms Price & Greenberg's hypothesis (1989) that neuro-

peptides sharing a high level of sequence identity with FMRFamide (i.e. the FXRFamides, where X is M, L or I) are restricted to the protostomian phyla.

The starfish peptides are clearly related themselves, for GFNSALMFamide is identical to the C-terminal

sequence of SGPYSFNSGLTFamide in all but three of

eight amino acid residues. Furthermore, the residues that differ represent relatively conservative substitutions at the level of amino acid structure. This also


(a)

S a

8

6 CO -2) 5- C S 4-

2

1

0

(c)

80

70

60

50 22

40

E 30

20 -

10 -

0- 0

s

A

L

M

1 2 3 4

cycle

. . !. - - .- II - I - - m

2

I -

I -

I -

1-

1-

I -




(b) 10

-s--c-- S --a- s

~-p -.--t- Y

-0-- N

G T L R* A A T

F

-e C=

Cd

8-

6-

4-

2-

9 1011 12131415

1275

[I(ldllUJIbl^l,i,)lSii( ,All,l,.iJ,.

1200 1300 m/z

(d) 100-

a80

a 40

C fl'. 20-

885-3

4 5 6 7 8

cycle 875 900 925

m/z

-*--- G

---- N - A

---o L

S

L

2 3 4 5

cycle

(f) 100

a) , 80

= 60

i 40

20

6 7 8

901-4

I; I Iii VI I ..1

890 900 910

m/z

Figure 4. Identification of the SALMFamides in Asterias forbesi by automated Edman degradation and fast atom bombardment mass spectrometry. (a), (c) and (e) show the PTH amino acids from Edman degradation on peaks D, E and C respectively. In (e), only a small amount of peptide was available for sequencing; glycine was the most abundant amino acid in each cycle but it was still possible to deduce a partial sequence (GFNSAL) from the data

by identifying the amino acid that increased in abundance at each cycle, compared to the previous cycle, and then decreased in abundance in the subsequent cycle. (b), (d) and (f) show the mass of the predominant molecular ion

(M + H)+ in fast atom bombardment mass spectra of peaks D, E and C respectively. The sequence GFNSAL is not consistent with the masses 885.3 or 901.4 but the mass of peak E (885.3) is consistent with the sequence Gly-Phe-Asn- Ser-Ala-Leu-Met-Phe-NH2. Peak C is 16 Daltons heavier than peak E so must be the oxidized form of GFNSALMFamide. Oxidation of the peptide molecule involves the reaction of a single oxygen atom with the sulphur atom in the side chain of the methionine residue. The sequence and mass (1275) identifies peak D as Ser-Gly-Pro- Tyr-Ser-Phe-Asn-Ser-Gly-Leu-Thr-Phe-NH2.,

extends to the nucleotide level where in all three cases

only single base changes could generate these substitutions (figure 5).

We propose that these two peptides are founder members of a new family of neuropeptides named the SALMFamides. This name is derived from the single letter amino acid notation for the pentapeptide that was isolated from A. rubens as a result of acid lysis of the native peptide, GFNSALMFamide. The name is con- venient for it is not biased towards any physiological role or phylogenetic distribution, it follows a system of

peptide nomenclature established by Price & Green-

berg (1977) and it is pronounceable (i.e. 'psalm- famides'). We shall refer to GFNSALMFamide and SGPYSFNSGLTFamide as SALMFamide 1 and 2 (S1 and S2) respectively and if other members of this

family are isolated in the future we recommend that this numbering system be continued.

The extensive sequence identity ofS1 and S2 suggests that the gene or genes that encode them evolved

through intragenic or genic duplication respectively. An example of the former is found in the snail Lymnaea stagnalis where FMRFamide and a related neuro-

peptide FLRFamide are encoded by a single gene. This


(a)

S

G s

400

350

300

250

200 Co

E m

100

50

0

cycle (c)

70

60

50

Co 40

g 30

20

10

G - F -- N - S -* A

----- L

s A

(e)

20- G

CO

S (1)

Pc

15

10




S1 Gly GGU GGC

-Phe-Asn-Ser -AlaC GCX

-Leu- -Met- AUG

?Phe-NH2

S2 Ser-Gly-Pro-Tyr-Ser Phe-Asn-Ser Gly Leu -Thr Phe-NH2 AGU GGX ACG AGC

Figure 5. Structures of the SALMFamides (SI and S2). They are clearly related since SI is identical to the C-terminal octapeptide of S2 in all but three amino acid residues. The three residues that differ in S1 and S2 are boxed with codons for each amino acid underneath. Single base changes (underlined) could generate these three amino acid substitutions.

gene encodes a precursor protein that contains nine copies of FMRFamide and two copies of FLRFamide; the multiple copies probably having evolved through repeated intragenic duplication (Linacre et al. 1990). The ratio of FMRFamide to FLRFamide in the gene is also reflected in the amounts of each peptide that can be detected in extracts of molluscan ganglia. About five times more FMRFamide than FLRFamide is detected when extracts are assayed by RIA after HPLC fractionation (Price et al. 1987). In starfish radial nerve cords, S1 and S2 are present in roughly equimolar amounts (figures 1 a and 2a) so a hypothetical SALMFamide gene might encode the same or a similar number of copies of each peptide. It remains equally likely, of course, that the two peptides are encoded by separate genes.

The function of the SALMFamides is as yet not fully understood. However, we are currently testing synthetic S and S2 on a number of starfish neuromuscular

preparations and preliminary results suggest that S2 plays a fundamental role in stomach relaxation (M. R.

Elphick & M. C. Thorndyke, unpublished observa-

tions). Identifying the neurons that express S1 and S2 will provide important clues to their function and we are currently raising both monoclonal and polyclonal antisera that are specific for S1 or S2 in order to map, immunocytochemically, their distribution in Asterias.

The SALMFamides are the first neuropeptides identified in species belonging to the phylum Echino- dermata. We have also detected S -like immuno-

reactivity in the nervous systems of other echinoderms such as brittlestars and sea cucumbers (M. R. Elphick & M. C. Thorndyke, unpublished observations). FMRFamide is common to all molluscs (Price et al.

1987) and similarly, the SALMFamide neuropeptides may be common to all echinoderms. Clearly the immunoreactive substances in other echinoderms will have to be identified in order to corroborate this prediction.

This work was done during the tenure of a Science and Engineering Research Council (U.K.) studentship to M. R. E. In addition, we acknowledge support from the Grass Foundation, National Institute of Health, U.S.A. (grant HL28440 to M.J. Greenberg), National Science Founda- tion, U.S.A. (grant DCB-8616356) to M. J. Greenberg) and Boehringer Ingelheim. We thank especially Dr M. J. Greenberg for his help and encouragement throughout the project and Karen Doble for her help in the lab. The

assistance of Zyg Podhorodecki and Lynn Milstead in preparing figures is greatly appreciated. Peptide sequencing by automated Edman degradation was performed by B. F. Parten of the Protein Chemistry Core Facility at the University of Florida, Gainesville; M. G. Pickering of the Cell Biology Laboratory at RHBNC and by J.R. C. at the Beckman Research Institute of the City of Hope. Molecular weight determination by Fast Atom Bombardment Mass Spectrometry was performed by T. D. Lee. Thanks also to Professor G.J. Dockray (University of Liverpool) for his helpful comments on the manuscript.

REFERENCES

Bewick, G. S., Price, D. A. & Cottrell, G. A. 1990 The fast response mediated by the C3 motoneurone of Helix is not attributable to the contained FMRFamide. J. exp. Biol. 148, 201-209.

Boer, H. H., Schot, L. P. C., Veenstra, J. A. & Reichelt, D. 1980 Immunocytochemical identification of neural ele- ments in the central nervous system of a snail, some insects, a fish and a mammal with an antiserum to the molluscan cardio-excitatory tetrapeptide FMRF-amide. Cell Tiss. Res. 213, 21-27.

Brussaard, A. B., Kits, K. S., & Ter Maat, A. 1989 One receptor type mediates two independent effects of FMRFa on neurosecretory cells of Lymnaea. Peptides 10, 289-297.

Chaet, A. B. 1964 A mechanism for obtaining mature gametes from starfish Biol. Bull. 126, 8-13.

Cottrell, G. A. 1989 The biology of the FMRFamide-series of peptides in molluscs with special reference to Helix. Comp. Biochem. Physiol. 93A, 41-45.

Elphick, M. R., Emson, R. H. & Thorndyke, M. C. 1989a FMRFamide-like immunoreactivity in the nervous system of the starfish Asterias rubens. Biol. Bull. 177, 141-145.

Elphick M. R., Price, D. A., Lee, T. D. & Thorndyke, M. C. 1989 b The SALMFamides: a new family of neuropeptides isolated from an echinoderm. Soc. Neurosci. Abstr. 15, 1276.

Grimmelikhuijzen, C. J. P., Dockray, G. J. & Schot, L. P. C. 1982 FMRFamide-like immunoreactivity in the nervous system of hydra. Histochemistry 73, 499-508.

Holmgren, S. 1989 The comparative physiology of regulatory peptides. London and New York, Chapman and Hall.

Linacre, A., Kellet, E., Saunders, S., Bright, K., Benjamin, P. R. & Burke, J. F. 1990 Cardioactive neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFamide) and novel related peptides are encoded in multiple copies by a single gene in the snail Lymnaea stagnalis. J. Neurosci. 10, 412-419.

Man-Son-Hing, H., Zoran, M.J., K. Lukowiak, K., & Haydon, P.J. 1989 A neuromodulator of synaptic transmission acts on the secretory apparatus as well as on ion channels. Nature, Lond. 341, 237-239.





O'Shea, M., & Schaffer, M. 1985 Neuropeptide function: the invertebrate contribution. Ann. Rev. Neurosci. 8, 171-198.

Price, D.A. 1982 The FMRFamide-like peptide of Helix aspersa. Comp. Biochem. Physiol. 72C, 325-328.

Price, D.A. & Greenberg, M.J. 1977 Structure of a molluscan cardioexcitatory neuropeptide. Science, Wash. 197, 670-671.

Price, D. A. & Greenberg, M.J. 1989 The hunting of the FaRPs: the distribution of FMRFamide-related peptides. Biol. Bull. 177, 198-205.

Price, D. A., Cottrell, G. A., Doble, K. E., Greenberg, M. J., Jorenby, W., Lehman, H. K. & Riehm, J. P. 1985 A novel FMRFamide-related peptide in Helix: pQDPFLRF- amide. Biol. Bull. 169, 256-266.

Price, D. A., Davies, N. W., Doble, K. E. & Greenberg, M. J. 1987 The variety and distribution of the FMRFamide- related peptides in molluscs. Zool. Sci. 4, 395-410.

Thorndyke, M. C. & Goldsworthy, G. J. 1988 Neurohormones in invertebrates. Cambridge: Cambridge University Press.

Received 24 September 1990; accepted 19 October 1990




the salmfamides: a new family of neuropeptides isolated from an echinoderm

Documents