Download FMRFamide, a Cardioexcitatory Neuropeptide of Molluscs: An Agent

AMER. ZOOL., 19:163-174 (1979).
FMRFamide, a Cardioexcitatory Neuropeptide of Molluscs: An Agent in
Search of a Mission
MICHAEL J. GREENBERC AND DAVID A. PRICE
Department of Biological Sciences, Florida State University, Tallahassee, Florida 32306
SYNOPSIS. Aqueous extracts of molluscan ganglia have cardioexcitatory activity that is not
attributable to the usual neurohumors: 5-hydroxytryptamine, acetylcholine or dopamine.
Morphological, physiological and biochemical studies have suggested that this cardioexcitation is caused by a handful of unidentified neurohormones, mostly polypeptides. One of
the active components of extracts of the pooled central ganglia of Macrocallista nimbosa
(Bivalvia) has now been isolated, purified, characterized and synthesized. This substance,
previously designated peak C, is a tetrapeptide amide: phenylalanyl-methionyl-arginylphenylalanine amide (Phe-Met-Arg-Phe-NH2; FMRFamide). Low concentrations of FMRFamide (threshold: 10~9-10~8M) induce rhythmical activity in isolated quiescent bivalve
hearts and augment the force and frequency of contraction of those already beating. The
peptide also produces contractures of non-cardiac molluscan muscles, including especially
the radula protractor of the whelk, Busycon contrarium, a sensitive assay object. The effects
of FMRFamide on Mercenaria mercenaria hearts appear to be mediated by adenosine 3',
5'-monophosphate (cycwic AMP). Peak C activity occurs in all species tested in all the major
molluscan classes; but we do not yet know whether all Peak C's are FMRFamide. That
FMRFamide is a neurosecretory product has still to be established, and its normal physiological role in molluscs has yet to be defined. Nevertheless, we suppose that, together with
other neuropeptides, FMRFamide is released into the hemolymph to provide long-term
maintenance and regulation of tone, rhythmicity and excitability of visceral muscle.
cology has been reviewed by Greenberg et
al.,
1973.) Intractably hypodynamic prepaThe most fundamental characteristic of
rations
frequently respond to an aqueous
cardiac muscle is its spontaneous rhythmiof
pooled ganglia.
extract
cal activity. The rhythmicity of the myoReports
that molluscan ganglion extracts
genic hearts of molluscs, tunicates, verteor contractions
produce
cardioexcitation
brates and insects, arises in the muscle
and
that these efof
non-cardiac
muscle,
itself and usually continues when the myocardium, or a portion of it, is suitably iso- fects are not attributable to the usual mollated in an appropriate saline medium. Yet, luscan neurohumors — acetylcholine (ACh),
in the course of surveying the pharmacol- 5HT, or dopamine—have been accumuogy of bivalve hearts (e.g., Greenberg, 1965, lating for about two decades; the observa1969), we have found that some prepara- tions come from species representing every
tions (and in some species, all preparations) molluscan Class except Scaphopoda (sumbecome arrhythmic in vitro. Among vari- marized by Greenberg et al., 1973). The
ous treatments, use of the molluscan car- unidentified excitor, at first referred to as
dioexcitatory neurotransmitter, 5-hy- "Substance X" (Hill and Welsh, 1966), was
droxytryptamine (5HT), often induces soon shown to be composed of from three
beating in quiescent hearts; often it does to five chromatographically separable ennot. (The alchemy of oscillogenic pharma- tities; the number depended upon the
particular species examined (Frontali et at,
1967; Agarwal et al., 1972). Three of the
This work was supported by NIH Grant HL-09283
to MichaelJ. Greenberg. This is Contribution No. 100 active agents (designated peaks A, B and
INTRODUCTION
from the Tallahassee, Sopchoppy and Gulf Coast C) from Mercenaria mercenaria ganglion
Marine Biological Association. We acknowledge the
help of Association members in collecting and dis- extracts proved to be peptides (Frontali et
secting the thousands of clams used in the studies al., 1967).
reported here.
Peak C, alone, has been detected in the
163
164
MICHAEL J. GREENBERG AND DAVID A. PRICE
nervous tissue extracts of every species
investigated (Greenberg et al., 1973).
Moreover, although it is highly concentrated in the ganglia, peak C activity is also
widely distributed in other tissues of the
dried ganglia dissected from 500 clams
yields about 5 nanomoles of FMRFamide;
i.e., the pooled ganglia from one clam contain about 10 picomoles.
FMRFamide was synthesized, and was
then purified by the same techniques used
bivalve, Macrocallistu nimbosa (Agarwal et
al., 1972). Finally, while all of the active in the purification of the natural peptide.
substances increase the force of contrac- Synthetic and natural FMRFamide gave
tion and frequency of normally beating identical thin-layer chromatograms. The
hearts, only peak C also induces beating in biological effects of the natural and synhypodynamic myocardia (Agarwal and thetic materials were qualitatively and quanGreenberg, 1969). Because of its ubiquity, titatively indistinguishable, whether they
distribution in molluscan tissues, and an- were tested on the clam heart or the radula
tiarrhythmic action, we began the process protractor (details of purification, characof identifying peak C. Recently we suc- terization and synthesis in Price and Greenberg, 1977a,6).
ceeded (Price and Greenberg, \977a,b).
In retrospect we can see that the purification
of FMRFamide was facilitated by
PURIFICATION AND CHARACTERIZATION
its composition. FMRFamide is a strong
Sun-ray venus clams, Macrocallista nim- base due to the guanidino group of its arbosa, were collected in batches of 800 to ginyl residue; but it is also non-polar due
1,000 (ten bushels) from St. Joseph Bay to its other three residues. Most substances,
near Apalachicola, Florida. The cerebral, including impurities, have only one or the
pedal and visceral ganglia were dissected other of these two properties. Therefore,
out, pooled, and lyophilized. The peak C by using separative procedures exploiting,
activity was purified by the combined use in turn, its basic and non-polar characterof solvent extractions, gel filtration and istics, FMRFamide could be isolated in relion-exchange chromatography. During atively pure form in a small number of
purification, activity was monitored with a steps.
parallel bioassay system consisting of the
radula protractor muscle of the whelk BusyPHARMACOLOCY
con contrarium, and the heart of the clam
Mercenaria mcrcenaria. T h e responses of
these preparations are discussed below.
Purified peak C has a phenylalanine-like
absorption spectrum and an isoelectric point
greater than pH 10. It is inactivated by
trypsin, chymotrypsin and Pronase, and
gives a positive reaction with both ninhydrin and the Sakaguchi reagent for arginine. Its amino acid composition is Phe2.oo
Metfl.mArg, 12. On the basis of N-terminal
analysis, Edman-dansyl degradation and
tryptic digestion, the structure was found
to be that of a tetrapeptide amide: PheMet-Arg-Phe-NH2. (We call the peptide
FMRFamide since F, M and R are the approved one-letter abbreviations for phenylalanine, methionine and arginine, respectively. The designation thus conveys both
the amino acid sequence and the substitution of the carboxy terminal. And it is a
euphonious partial acronym.) One gram of
The parallel bioassay
The Mercenaria heart and the Busycon
radula protractor muscle are both classical
assay objects whose isolation, preparation
and pharmacological characteristics have
long been studied (Welsh and Taub, 1948;
Hill, 1958).
The effect of FMRFamide on the radula
protractor is a sustained contraction; threshold is 1-3 x 10"9 M. ACh, the presumed
excitatory neurotransmitter, has the same
effect, but is antagonized by prior treatment with benzoquinonium chloride (BQ).
FMRFamide contractures are not affected
by BQ. Other indolealkylamines, as well as
5 HT, and catecholamines either relax the
protractor muscle or have no effect; the
particular response depends upon the
resting tension. If a protractor is in ACh
contracture, addition of 5HT relaxes the
NEUROPEPTIDE CARDIOEXCITATION IN MOLLUSCS
muscle and induces rhythmical contractions (Hill, 1958). FMRFamide contractures are reduced in the presence of 5HT.
FMRFamide increases the amplitude of
beat of active Mevcenaria hearts and induces
beating in quiescent preparations; threshold is 1-2 x 10~8 M. The effect is similar,
qualitatively and quantitatively to that of
5HT, but is not blocked by the 5HT antagonist, methysergide (UML). In contrast
to its effect on the radula protractor, ACh
inhibits the beat of the Mercenaria heart,
and even very low concentrations (10~ 9 10~8 M) cause arrest.
Since FMRFamide excites both the radula
protractor and the clam heart, and since
these effects are not blocked by either of
the appropriate inhibitors, BQ or UML, in
neither preparation can the peptide be acting by releasing 5HTor ACh.
A set of tetra-r tri- and dipeptide analogs
of FMRFamide has been synthesized; all
contain various permutations of the same
three amino acids as are found in the parent compound. These peptides, the constituent amino acids themselves, and three
additional arginine derivatives (benzoyl
arginine, benzoyl arginine amide and tosyl
arginine amide), were all tested on the
protractor muscle. Of all these compounds,
only Phe-Met-Arg-Phe-OH and Met-ArgPhe-NH2 had any activity at all; and the
relative potencies were no better than, respectively, 1/100 and 1/1000 that of FMRFamide (Table 1). Thus, the biological acTABLE 1. Structure-activity relations of FMRFamide
on Busycon contrarium radula protractor."
Peptide
Phe-Met-Arg-Phe-NH.,
(FMRFamide)
Phe-Met-Arg-Phe-OH
Phe-Met-Phe-Arg-NH.,
Phe-Met-Phe-Arg-OH
Mei-Arg-Phe-NH2
Phe-Met-Arg-NH.,
Arg-Phe-NH.,
Phe-Arg-OH
Met-Arg-NH2
d
Relative potency"
1.00
.01
c
—
.001
—
—
—
—
Data from Price and Greenberg, 1977a.
Relative effect of a threshold dose of FMRFamide
(3x10 » M ) = 1.00.
'• No effect up to, and including, 10"' M.
11
165
tivity of FMRFamide appears to depend on
the arrangement of its four constituent
amino acid residues in the specific sequence
determined.
Possible mechanisms of FMRFamide
cardioexcitation
The effect of FMRFamide on the electrical properties of the molluscan myocardium have been examined only in a preliminary experiment. Partially purified
Macrocallista peak C induced regular, rhythmic spiking and mechanical activity in quiescent Modiolus hearts; the membrane
potential was not affected (Wilkens and
Greenberg, 1973). The ability of thyrotropin-releasing hormone (TRH) to induce
repetitive calcium spikes in cultured
anterior pituitary cells (Taraskevich and
Douglas, 1977) is in striking parallel with
the effect of FMRFamide on bivalve heart
muscle.
Previous studies in our laboratory had
suggested that the cardioexcitatory action
of 5HT on the heart of Mercenaria was
mediated by cyclic AMP (Higgins, 1974;
Higgins and Greenberg, 1974). Recently,
therefore, we returned to consider the effect of FMRFamide on adenylate cyclase
activity and cyclic AMP concentration in
Mercenaria ventricles, and to compare these
effects to those of 5HT (Higgins et al.,
1978).
In summary, hearts treated with either
5HT or FMRFamide showed an increase
in adenylate cyclase activity and an elevation in cyclic AMP concentration. These
effects were dependent on dose and the
duration of treatment. The rate of increase
of cyclic AMP concentration is as fast as the
rate of development of the positive inotropic effect. While the effect of 5HT is
blocked by UML, that of FMRFamide is
not; thus these two substances act at different receptor sites.
The independence of action of 5HT and
FMRFamide was recently pursued by Higgins (1977), starting with the old observation (Greenberg, 1960) that high concentrations of 5HT first excite the isolated
ventricle of Mercenaria and then specifically desensitize it to further additions of
166
MICHAEL J. GREENBERG AND DAVID A. PRICE
the neurohumor. Higgins demonstrated
that this 5HT-induced tachyphylaxis includes both the desensitization of the myocardial adenylate cyclase and a reduced
increase in intracellular cyclic AMP. However, FMRFamide can still increase the
contractility, adenylate cyclase activity and
cyclic AMP concentration of a ventricle
that is tachyphylactic to 5HT. Thus, while
FMRFamide and 5HT appear to share a
common mechanism of action, the two
agonist-receptor interactions initiating this
mechanism, must occur at different sites
on the cell membrane.
The actions of FMRFamide on other molluscan
muscles
Recently, we have been surveying the
effects of FMRFamide on selected molluscan muscle preparations. First, the isolated
hearts from a number of species of bivalves, including Mytilus edulis, Chione cancellata, Crassostrea virginica and Modiolus
demissus, were tested. Invariably, the effect
of FMRFamide is excitatory: an increase in
amplitude, frequency or both. That this
response should also be characteristic of all
M. demissus hearts is of interest since 5HT
may depress or excite individual preparations from this species (Fig. 1 A). Thus, the
mimicry of 5HT by FMRFamide on molluscan myocardium is not general. Furthermore, the adenylate cyclase activity of M.
demissus hearts is very low (Higgins, 1974).
Consequently, FMRFamide excitation may
be mediated by other intracellular mechanisms; certainly, this seems to be occurring
in the case of Modiolus heart.
FMRFamide causes the anterior byssus
retractor muscle (ABRM) of Mytilus edulis
to go into "catch." That is, a suitable dose
of the peptide produces a sustained contracture, and the muscle relaxes only slowly
even after the FMRFamide is washed out
of the bath. The rate of relaxation is markedly increased by 5HT (Fig. I B). Thus, the
effect of FMRFamide on the ABRM, as on
the radula protractor, resembles that of ACh
rather than 5HT. It follows that, since the
effect of 5HT on the ABRM is apparently
mediated by cyclic AMP (reviewed by
Twarog, 1976), the mechanism of action
of FMRFamide on this muscle must be different.
Finally, two visceral muscles have been
challenged with FMRFamide: the rectum
of Macrocallista, and the esophagus of the
prosobranch gastropod, Fasciolaria tulipa.
FMRFamide induces rhythmic activity in
both muscles; the response is especially
marked in the esophagus (Fig. 1C). Again,
5HT cannot induce rhythmicity in a quiescent preparation, and actually inhibits such
activity when it is present (Fig. 1C).
In summary, while 5HT and ACh are
notable for the variability of their pharmacological effects {e.g., Greenberg, 1969),
FMRFamide appears to be uniformly excitatory. However, other mechanisms, in
addition to augmentation of adenylate cyclase activity, will probably be found to underlie these effects.
Finally, FMRFamide has been dutifully
tested on isolated cardiac and vascular
muscle of rat (Price and Lipner, personal
communication) and guinea pig ileum (Higgins, personal communication). Such small
effects as were observed, occurred only at
very high concentrations. Conversely, we
have also tested, on the Mercenaria heart
and the Busycon radula protractor: Substance P, eledoisin, physalaemin, oxytocin,
vasopressin and the C-terminal tetrapeptide of gastrin. All were inactive in bath
concentrations up to 0.1 mM.
DISTRIBUTION
Are all peak C's FMRFamide?
The identity of all peak C's follows from
the observation that: if the pooled ganglia
of any mollusc are extracted and purified
according to a defined protocol, a predictable, limited elution volume from the final
get filtration will produce a characteristic
excitation, including the induction of rhythmicity, when assayed on the isolated heart
of a venerid bivalve (Agarwal et «/., 1972).
The number and diversity of molluscs already tested is sufficiently large, and the
procedure leading to the result is sufficiently involved, that mere coincidence
appears unlikely. However, faced with the
apparent difference between the egg-lay-
B
t
F
°8
5xlO"°M
5HT IO"6M
FIG. I. Effects of FMRFainide (Fa) and 5-hydroxytryptamine (5HT) on isolated molluscan muscles.
Drugs are added to the supervision medium at the
upward-pointing arrows, and doses are expressed as
concentrations in the organ bath. A) Ventricles of
Modiolus demissus. Upper and lower records are from
different preparations. The baths were washed out
after the 5HT response and before the addition of
FMRFamide. B) Anterior byssus retractor muscle
(ABRM) of Mytilus edulis. Downward-pointing arrow
Fa IO"8M
indicates washout of the organ bath. C) Esophagus of
Fasciolaria tulipa. Upper and lower records are from
the same preparation; doses of 5HT and FMRFamide
were repeated. The bath was washed after the first
5HT response; but the second dose of FMRFamide
was not washed out before 5HT was added. Time
bar: I min. Temperature: A, C, 2I°C; B, 16°C.
(Price, Painter, Klausman and Greenberg, unpublished observations.)
168
MICHAELJ. GREENBERG AND DAVID A. PRICE
ing hormones of Aplysia and Pleurobran- FMRFamide were the product of such a
chaea (Ram et al., 1977), a healthy skepti- process, then peaks A or B, or the molluscism about the identity of all peak C's with can reproductive hormones, might be deFMRFamide is essential until the identity rived from the same large precursor. Howhas been conclusively demonstrated.
ever, in the absence of any corroborative,
especially chemical, data, further speculation would be gratuitous.
FMRFamide and other molluscan
neuropeptides might be homologous
A number of molluscan neuropeptides
have, by now, been identified, but not chemically characterized (Table 2; reviewed by
Frontali and Gainer, 1977). Their known
effects are defined by their assays and therefore reflect, primarily, the interests of the
laboratories where they are studied. However, by analogy with both crustacean (e.g.,
Mordue and Stone, 1977) and vertebrate
(reviewed by Wallis, 1975) hormones, any
of these peptides could have additional actions on other systems in the same animal,
or in other molluscs. The acquisition of new
functions in different molluscan groups
could have been accompanied by relatively
small changes in the amino acid composition. Consequently, FMRFamide might be
homologous, or even identical, to one or
more of the factors, all chemically uncharacterized, listed in Table 2. But is it?
Data by which the molluscan peptides
might be chemically distinguished comprises
a scattering of sensitivities to proteolytic
enzymes and some molecular weights obtained from gel filtration (Table 2). On the
basis of their lack of sensitivity to trypsin
and chymotrypsin, neither the cardioexcitatory neurosecretory substance (NS)
from cephalopod vena cava, nor the various bursting pacemaker potential (BPP)
inducers isolated from extracts of gastropod ganglia or their constituent cells
(references in Table 2), are likely to be related to FMRFamide. Since it co-chromatographs with peak C, FMRFamide
cannot be identical with peaks A or B. Moreover, the molecular weight proposed for
the reproductive neuropeptides is about
ten times higher than that of FMRFamide
(Table 2). However, many hormones, including neuropeptides, may be produced
by the action of specific proteolytic enzymes on high molecular weight precursors (reviewed by Gainer et al., 1977). If
The possibility that FMRFamide occurs and is
functional in other phyla
The possible polyphyletic distribution of
FMRFamide is best considered as part of
the larger problem: the degree of homology between the arrays of neuropeptides
characteristic of each phylum. A definitive
solution would require that the chemical
structures of at least most of the neuropeptides in three or four phyla be known.
In fact, they are mostly unknown except in
the class Vertebrata where between 15 and
20 neuropeptides have been chemically
identified and localized (references in Zettler, 1976; Brownstein, 1977; Robberecht
et al., 1978). In contrast, of about 30 arthropod neuropeptides listed by Frontali
and Gainer (1977), only 4 are chemically
identified (Fernlund and Josefsson, 1972;
Stone rf al., 1976; Fernlund, 1976; Brown,
1975; Brown and Starratt, 1975; Starratt
and Brown, 1975). And in the molluscs,
only about 13 neuropeptides have so far
been characterized (Table 2); some of these
may be chemical homologs, but only FMRFamide is known.
Even with the paucity of information, we
can still make the argument that there is
little or no homology between the neuropeptides of evolutionarily disparate phyla.
Firstly, there is little chemical similarity
between FMRFamide, any of the known
arthropod neuropeptides, and those of
vertebrates. Kleinholz (1976), for example,
noted the dissimilarity between the distal
retinal pigment hormone (DRPH) and the
seven amino acid sequence required for
vertebrate peptides to have MSH-like activity. Secondly, in many instances where it
has been sought, cross-reactivity has not
been found. This was our experience, described above, with FMRFamide and a number of vertebrate peptides. For another
example, the absence of enkephulin acthity
NEUROPEPTIDE CAROIOEXCITATION IN MOLLUSCS
and opiate receptor binding in the nervous
systems of clams, as well as various types of
arthropods and planarians, has been shown
(Simantov et al., 1976).
Nevertheless, the following evidence suggests that the notion of lack of homology
should be viewed with caution. Firstly, two
vertebrate neuropeptides have been found
in molluscs. Immunoreactive gastrin, recently demonstrated in vertebrate brain
(Vanderhaeghen et al., 1975), also occurs
in gastropod blood and gut — but not brain
(Straus et al., 1975). And immunoreactive
TRH was located in the circumesophageal
ganglia of gastropods (Grimm-j0rgensen et
al., 1975). However, these identifications,
based only on radioimmunoassay, should
be confirmed by standard biological analyses {e.g., Skelley^a/., 1973).
Secondly, eledoisin, a toxin occurring in
the posterior salivary glands of cephalopods, is an analog of the vertebrate neuropeptide, Substance P; both are undecapeptides with similar carboxy terminals:
-Phe-(Ile or Phe)-Gly-Leu-Met-NH2 {e.g.,
Leeman et al., 1977). That molluscan tissues can manufacture a Substance P-like
peptide is provocative; but eledoisin has
not yet been found in molluscan nerve
cells.
Finally, low concentrations of vasopressin and oxytocin initiate bursting pacemaker potential (BPP) activity in particular
cells of gastropod ganglia (reviewed by
Barker, 1977; Levitan and Treistman,
1977), and increase cyclic AMP levels in
Helix ganglion homogenates (Levitan and
Treistman, 1977). In these effects, the
neurohypophysial hormones are mimicking the actions of peptides which occur in
crude ganglion extracts of these species
(Table 2). The endogenous peptides are
not oxytocin or vasopressin (Ifshin et al.,
1975; supported by data of Levitan and
Treistman, 1977). Moreover, neither oxytocin nor vasopressin has been demonstrated in molluscan tissues.
PHYSIOLOGICAL ROLE
Is F'MRFamide
a product oj' neurosecretion?
We have been assuming throughout this
169
discussion that, since FMRFamide (or peak
C) is concentrated in nervous tissue, it is
probably a neurosecretory product, stored
in membrane vesicles and released, either
from the ganglia, or from peripheral sites
of innervation, particularly the heart. This
notion is supported primarily by cytological observations; but some experimental
evidence can also be adduced.
Firstly, the cardiac innervation in a number of pulmonate gastropods has been examined by transmission electron microscopy (Helix pomatia: Cottrell and Osborne,
1969; four species of achatinids: Nisbet and
Plummer, 1966; 1969; Helisoma tenue and
Helix aspersa: Simpson, 1969; Lymnaea stag-
nalis: Plesch, 1977). These investigations
showed, in sum, that only the auricles of
pulmonate hearts are heavily innervated
and especially in the region close to the auriculo-ventricular junction. Furthermore,
many of the axons in this region terminate
adjacent to the cardiac lumen. Visible in at
least some of the nerve terminals are uniformly granulated vesicles (100-250 nra)
which, on the basis of fluorescence microscopy, contain neither 5HT nor dopamine.
The consensus is that these are neurosecretory granules. Cottrell and Osborne
(1969) suggested that the auriculo-ventricular junctions function as neurohemal
organs; their notion was supported by Nisbet and Plummer (1969); and Plesch (1977)
found axon terminals in Lymnaea heart
containing granules similar to those in
particular neurosecretory cells of the visceral and parietal ganglia. Recently, large,
opaque vesicles were also observed in some
nerve endings in the auriculo-ventricular
region of the heart of the clam, Katelysia
rhytiphora (Sathananthan and Burnstock,
1976). That these vesicles do not contain
5HT, ACh or dopamine was demonstrated;
but the proposal that they release an ATPlike agent remains problematical.
Crude extracts of snail auricles do, in
fact, have cardioexcitor activity (reviewed
by Greenberg et al., 1973); and extracts of
Strophocheilus oblongus auricles (more than
ventricles) induce rhythmical activity in isolated penis retractor muscles of this pulmonate (Jaeger, 1966). Finally, Cottrell
(1966) and Cottrell and Maser (1967) car-
TABLE 2. Molluscan neuropeptides through 1977.
Designation
HEART AND
MUSCLE
FMRFamide
Peak Ca
PeakB
Source"
Pooled
ganglia
(M
(Macrocallista
nimbosa)
Pooled
molluscan
ganglia and
heart
Assay6
Effect"
NS
MW
(daltons)
597.75
Bivalve
Cardioexcitaheart;
tion; muscle
Busycon
contraction;
adenylate cyradula protractor m. 1 clase stimulation (only)
FMRFamide)
Isolated heart Cardioexci(Merce• tation.
nana)
Reference
Price and
Greenberg,
1977*
< 1,500
Frontali
et. al., 1967
< 1,500
Frontali and
Gainer, 1977
> 1,500
Peak A „
F II
Effect of proteolytic enzymes0
ChymoTrypsin trypsin Pepsin Pronase Papain
CEG (Stro- Isolated ven- Cardioexciphocheilus
tricle (Strotation.
oblongus)
phocheilus)
Isolated sysLong-lasting
Vena cava
ant NS
temic heart
cardioexcisystem
(Octopus)
tation.
(Cephalopoda)
NERVOUS ACTIVITY
PE
CEG (Helve Neurone Fl
pomatia)
(Helix,
RPaG)
Long-duration
BPP activn;
cAMP accum; adenylate cyclase
stimulation.
CEG (Otala Cell 11 (Otala, Long-duration
lactea)
RPaG)
BPP activn.
Agarwal,
etal., 1972
700-1,500 Hampe,
etal., 1969
-1,300
Blanchi
etal., 1973
700-5,000 Treistman
and Levitan, 1976
Levitan
and Treistman, 1977
700-5,000 Ifshin et al.,
1975
Bag cells
(Aplysia
AbG)
R 15 , L 1 0 (via
I
BPP induced &
L3 & RB)
augmented;
(Aplysia AbG) { EPSP (not IPSP)
ampl.increased.
Cell 11 (Otala) Long-duration
BPP activated.
[-
Mayeri and
Simon,
1975
Barker,
1977
700-5,000 Ifshin
etal., 1975
SALT AND WATER
BALANCE
—
/?is (Aplysia
AbG)
REPRODUCTION
Egg-laying
hormone
Bag cells
(Aplysia
AbG)
Egg-laying
hormone
PeG medial
lobe
(Pleurobranchaea
califor-
nica)
Egg capsule-laying PaOCPIG
(Busycon
substance
canaliculatum;
B. carica)
n
Injection into Osmotically
hemocoel
independent
(Aplysia
weight
californica)
increase.
1,500-30,000 Kupfermann
and Weiss,
1976
Injection into Egg-laying;
mature
long-term
Aplysia
behavorial
modification
6,000
Injection into Egg-laying;
mature
long-term
Pleurostereotyped
branchaea
behavior.
Injection into Laying unfilled
mature
egg capsules.
female
Toevs and
Brackenbury,
1969;
Arch, 1976
Arch and
Smock,
1977
4,000-7,000 Ram etal.,
1977
Ram, 1977
Busycon
All peak C's have not been shown to be FMRFamide.
Abbreviations: AbG (abdominal, or parietovisceral, ganglion); BPP (bursting pacemaker potential); CEG (circumesophageal ganglion or ring);
CP1G (cerebropleural ganglion); EPSP (excitatory postsynaptic potential); IPSP (inhibitory PSP); NS (neurosecretory); PaG (partial ganglion); PeG
(pedal ganglion); R15, L10, C, RB (identified cells in Aplysia AbG).
c
(+) enzyme destroys biological activity, (+ + , + + +) indicates relative effectiveness; (-) tested, but not effective; no entry: not tested.
b
172
MICHAEL J. GREENBERG AND DAVID A. PRICE
ried out a subcellular fractionation of Mercenaria ganglia and found a peak of cardioexcitatory activity, distinguishable from
that of 5HT, and associated with uniformly
granulated vesicles 100-300 nm in diameter. Since Cottrell's studies were completed
before Substance X was resolved into its
component activity peaks, we cannot be sure
that FMRFamide was one of the agents associated with the observed vesicles.
At present, about 10 cardioexcitor substances, including peptides and non-peptides, have been extracted from molluscan
nervous tissues and characterized (Table 2;
Frontali and Gainer, 1977; Agarwal et al.,
1972). At the same time, and considering
only pulmonates, about 10 or 11 kinds of
neurosecretory cells have been distinguished
by cytological techniques in the central ganglia (e.g., Boer et al., 1977; comparative review by Boer and Joosse, 1975); and there
are about 7 types in peripheral structures,
two of them in the heart (Plesch, 1977;
Lymnaea). In no instance has any characterized cardioexcitory peptide been associated unequivocally with a particular neurosecretory vesicle, either central or peripheral.
In summary, the status of FMRFamide
as a neurosecretory product, while probable, is unproven.
Possible functions
J. H. Welsh suggested that neurosecreted
polypeptides might act as long-duration,
long-range mimics of the short-acting neurotransmitters (Welsh, 1955). Since FMRFamide and 5HT have similar effects on the
heart of Mercenaria mercenaria, and since
the two substances share a common intracellular mechanism of action mediated by
an increased concentration of cyclic AMP
(Higgins, 1977; Higgins et al., 1978), this
system would appear to be a paradigm of
Welsh's proposal. However, even our preliminary survey of FMRFamide pharmacology points to an essential difference between its actions and those of 5HT and the
other neurotransmitters: FMRFamide lacks
the versatility, characteristic of neurohumors; its effect is always excitatory. In
that e\ent, the amount of FMRFainide re-
leased into the circulating hemolymph, or
possibly delivered by neural transport to
peripheral sites, would determine the general level of excitability of visceral muscles
in a mollusc, and this might be its role.
However, we have been focussing on
FMRFamide's stimulation of muscle merely
because this is our method of assay. As discussed above, analogous evidence from
other phyla implies that FMRFamide or a
homolog might have additional targets in
molluscs. Therefore, the physiological role
of FMRFamide must remain obscure until
the full range of its effects, its distribution
within the organism and its variation with
the condition of the animal — all presently
unknown — have been investigated.
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