Camp. iii&em. Physiol.Vol. 102C, No. 3, pp. 509-516, 1992
0306-4492/92 $5.00 + 0.00 Q 1992 Pergamon Press Ltd
Printed in Great Britain
ACTIONS OF APGW-AMIDE AND GW-AMIDE ON IDENTIFIED CENTRAL NEURONS OF THE SNAIL, HELIX ASPERSA M. L. CHEN and R. J. WALKER Department
of Physiology and Pharmacology, University of Southampton, Bassett Crescent East, Southampton, SO9 3TU, UK (Tel.: (0703) 594348 (Fax: (0703) 594319) (Received 17 October 1991)
Abstract-l. Actions of APGWamide and GWamide have been examined on identified central neurons of the snail, Helix aspersa. 2. In Fl neurons, both APGWamide and GWamide, 0.5-5 PM, reversibly inhibited the amplitude of evoked-IPSPs which were dopaminergic, but had no direct effect on membrane potential, cell firing rate, or dopamine-induced responses. These results indicate that the actions of APGWamide and GWamide in Fl neurons are at presynaptic sites. 3. At concentrations between 0.5 and lOpM, APGWamide and GWamide had direct postsynaptic effects on F2 neurons. They inhibited the spike activity and hyperpolarized the membrane potential of F2 neurons in a dose-dependent manner with a reversal potential around -85 mV which is close to E, 4. In K+ free solution, the inhibitory effects of APGWamide and GWamide were potentiated, while they were reduced by increasing external K+ concentration. Either tetraethylammonium (10mM) or 4-aminopyridine (500 PM) only partially prevented the inhibition induced by APGWamide and GWamide on F2 neurons. Combination of TEA (5 mM) and 4-AP (250 PM) could abolish this inhibition. However, neither 1 mM La*+ nor 10 mM Co2+ could prevent the inhibitory action of APGWamide and GWamide. This evidence indicates that the postsynaptic inhibition of APGWamide and GWamide on F2 neurons is due to an increase in K+ conductance and that both transient K channel (IA) and delay K+ channel (IK) were affected by these peptides. 4. APGWamide and GWamide exert both presynaptic and postsynaptic effects of Helix neurons,
depending on the neuron under study. They are qualitatively and quantitatively similar in their presynaptic or postsynaptic actions. We suggest that the amino acid sequence, Gly-Trp, might be essential to their neurobiological activity.
INTRODUCTION
A number of arthropod neuropeptides have been identified since 1974. The first one was the crustacean red-pigment-concentrating hormone (RPCH), isolated from shrimps (Femlund, 1974). Adipokinetic hormone (AKH) was isolated from locusts and its primary structure is closely related to RPCH (Stone et al., 1976). RPCH and AKH have biological actions on arthropod and molluscan tissues. The locust AKH and the crustacean RPCH have excitatory effects on the heart of the clam Mercenaria mercenuria (Greenberg et al., 1985). RPCH also shows modulatory actions on contractions of molluscan muscles (Yanagawa et al., 1988). Recently, a tetrapeptide, APGWamide, has been isolated from the ganglia of the prosobranch mollusc Fusinus ferrugineus and the ganglia of African giant snail, Achatina fulica Ferussac (Kuroki et al., 1990; Liu et al., 1991). The primary structure of APGWamide is related to the C-terminal tetrapeptide fragment of RPCH (Kuroki er al., 1990). This peptide was found to have a presynaptic modulatory action on contractions in various molluscan muscles (Kobayashi and Muneoka, 1990) and a postsynaptic inhibitory action on Achatina neurons, due to an increase in K+ conductance (Liu et al., 1991). However, the type of K+ channel involved in the neuronal modulation is unknown. The aim of this study is to elucidate the mode of action of APGW-
amide on the central neurons of Helix aspersa and to compare its actions with another synthetic peptide GWamide (H-Gly-Trp-NH,), as part of a structure-activity study. METHODS AND
MATERIALS
Experiments were performed on identified neurons of suboesophageal ganglia of the snail, Helix ospersa (Kerkut et al., 1975). The isolated ganglia were pinned on a small Sylgard-coated chamber with 1 ml volume and were immersed in normal saline which contained the following components (mM): NaCl 100, KC1 4, CaCl, 7, MgCl, 5, Tris base 5, final pH 7.5. Intracellular recordings were made using glass microelectrodes filled with 2 molar potassium acetate and with a resistance of 5-10 MR. The electrode was connected by a Ag-AgCl wire to a Dagan single electrode voltage-clamp system in the current clamp mode. Signals were displayed on a Tektronix 5111A oscillosope and permanent traces made using a Hewlett Packard 7402A chart recorder. In some experiments, the right pallial nerve trunk was sucked into a plastic suction electrode and stimulus pulses were produced by a Grass D9 stimulator. The duration of the stimulus pulse was between 0.5 and 2 msec, but the intensity of stimulation was varied. Application of chemicals was either by bath perfusion or by pressure ejection through a picospritzer II pressure ejector. The inner diameter of the tip of ejection electrodes is 2-3 pm. During the experiments, saline was continuously perfused over the preparation with perfusion rate 5% ml per min. In Na-free saline, sodium was replaced by equal 509
M. L. CHEN and R.J.
510
molar Tris base,CaClz 2 mM, MgC1, 10 mM, KC1 4 mM, using 12 molar HCI to adjust pH to 7.5. APGWamide: a gift from Professor Y. Muneoka (Hiroshima, University, Japan) GWamide: purchased from Bachem Feinchmikalien AG/Switzerland. RESULTS
In Fl neurons, 5 PM acetylcholine (ACh) depolarized the membrane and increased the cell firing rate. The ACh-induced response was sodium dependent, since it was abolished when Na’ was replaced by equal molar Tris in normal saline (Fig. IA). Bath application of APGWamide and GWamide (0.5, 1 PM) had no direct effect either on the membrane potential or on the cell firing rate of Fl neurons. However, at IOpM, both peptides slightly decreased cell firing rate (Fig. lB,C). An inhibitory postsynaptic potential (IPSP) could be evoked in Fl neurons when the right pallial nerve trunk was stimulated by a suction electrode (Fig. 2A. control). These evoked-IPSPs are dopaminergic and due to an increase in potassium conductance (Kerkut et al., 1969). When the amplitude of evoked-IPSPs was taken as 100% in normal saline, application of APGWamide 0.1, 0.5 and I PM for 2 min, reduced the amplitude of evoked-IPSP to 95.0 f 9.8%, 62.7 * 7.2%, and 38.3 -t_ 11.2% respectively of control (n = 4, f SEM) (Fig. 2A, Fig. 3). After washing with peptide-free saline for IOmin, the amplitude of evoked-IPSPs could be partially restored to 88.5 k 3.3% of control (n = 4, k SEM). GWamide also showed reversible inhibition on the evokedIPSPs in the same Fl neuron (Fig. 2B). In the presence of GWamide 0.1, 0.5 and 1 PM for 2 min, the amplitude of evoked-IPSPs was decreased to
Normal
Na
Saline
+AKWamide
0.5 JIM
tAPGWamide 1 p14
+AF%Wamide 10 J.IM
WALKEK
77.6 + 8.5%, 60.3 _t 9.7%, and 37.3 + 2.5% respectively of control (n = 4-5, + SEM). Following washing, the IPSPs returned to 90.7 _+9.3% of control (n = 4, + SEM, Fig. 3). A hyperpolarization could be produced by pressure ejection of dopamine (100 mM, 40 psi, 200 msec; Fig. 4A-1). In the presence of APGWamide and GWamide (I PM, 2 min), the amplitude of evokedIPSPs was reduced but the dopamine-induced responses were still present (Fig. 4A-2, 3). After washing with peptide-free saline, the amplitude of evoked-IPSPs was restored (Fig. 4A-4). In the presence of peptides, some synaptic potentials appeared in the period of dopamine-induced responses, so the actions of peptides in high Mg2+, low Ca2+ saline (20mMMgZ+,0.5mmCa 2+, Fig. 4B) were examined. In the presence of this saline, synaptic activity of presynaptic neurons could be abolished and so the direct effect of peptides on the identified neurons could be examined under these conditions. The dopamine-induced responses were not affected by either APGWamide or GWamide (I PM, 2 min, Fig. 4B2, 3). APGWamide and GWamide, at concentrations between 0.5 and 1 PM, showed dose-dependent inhibition on evoked-IPSPs in Fl neurons, but they had no direct effect on membrane potentials, cell firing rate or dopamine-induced responses. These results indicated that the actions of APGWamide and GWamide are on presynaptic sites. In contrast to Fl neurons, APGWamide and GWamide showed direct inhibitory effects on F2 neurons. In normal saline, pressure ejections of ACh (1 mM, 40 psi, 10 msec) could produce a depolarization (Fig. 5A). However, in the presence of Na+ free saline, ACh still evoked either a depolarization or hyperpolarization in F2 neurons, depending on the
saline
free
+Wash
t
t GWamide 0.5 uM
(GWamide 1 p4
twash
+
4 GWamide 10 ,uM
Fig. 1. Effects of APGWamide and GWamide on the spontaneous spike activity of Fl neuron in the suboesophageal ganglia of Hdiu asprrscr. (A) In normal saline, bath application of 5 PM ACh depolarized the neuron and increased the spike activity, whereas in the Na+ free solution, the ACh-induced response disappeared. Uparrows (t) indjcate when hrugs were bath applied and downarrows (1) show when drugs were washed bvi normal saline. (B) _ / Shows responses to 0.5, 1.O. 10 PM APGWamide. (C) Shows responses to 0.5, 1.0, 10 pM GWamide. All the intracellular recordings are from the same Fl neuron, resting membrane potential about -6OmV. The interval of peptide application is 1Omin.
Effect of
A
B
Control
Control
APGW-amide and GW-amide on snail neurons
APGWamide 0.1 t.LM
0.5
1.0
uM
Wash
n&f
aamide 0.1
Wash
j.l&l
30 mv --I 20 s Fig. 2. Effects of APGWamide and GWamide on the amplitude indicate that an IPSP could be evoked when the right pallial nerve ms, 4 V) through a plastic suction electrode. (A) Shows responses APGWamide, 0.1, 0.5, 1.0 FM, for 2 min, and then washing with Shows the actions of GWamide on the evoked-IPSPs from the potential about - 50 mV. The interval of peptide
membrane potential levels (Fig. 5B). The AChinduced responses on F2 neurons are chloride dependent and have a reversal potential of -35/-40mV (Kerkut and Meech, 1966). Both APGWamide and GWamide (0.5, 1, 5 PM) blocked cell firing and hyperpolarized the membrane potential in a dose-dependent manner of F2 neuron, (Fig. 5 CD). The reversal potential for this inhibitory event is around - 85 mV, close to E, in Helix neurons (Kostyuk, 1968; Bokisch and Walker 1986). The extent of membrane hyperpolarization induced by APGWamide (5 KM) on F2 neuron was affected by changing the external K+ concentration. In normal saline, the external potassium concentration is 4 mM and 5 ~1M APGWamide could produce hyperpolarizing responses of 16.7 + 1.OmV (n = 3 + SEM; Fig. 6B). In K+-free saline, the hyperpolarization induced by peptide was increased to 23.3 &-1.0 mV (n = 3, +_SEM; Fig. 6A). The amplitude of the hyperpolarization was reduced by increasing external K+ concentration (Fig. 6 C,D,E). The relationship between membrane hyperpolarization induced by APGWamide and GWamide (5 PM) and external K+ concentration is shown in Fig. 7. Fig. 8A shows that inhibition was induced by both 5,~cM of APGWamide and GWamide in normal saline. In the presence of either 10 mM tetraethyl ammonium (TEA) or 500 p M 4-aminopyridine (4AP), the inhibitory actions could be partially blocked by either K+ channel blocker (Fig. 8B,C). However, a combination of 5 mM TEA and 250pM 4-AP abolished the inhibition induced by peptides in F2 neurons (Fig. 8D). After washing with normal saline, the inhibition induced by either peptide could be restored, Fig. 8E shows the recovery to APGWamide (5pM). Furthermore, we also found that neither 10 mM Co’+ nor 1 mM La’+ could block the inhibition induced by both APGWamide and GWamide (5 p M) on F2 neurons.
of evoked-IPSPs of Fl neuron. Dots was stimulated by a square pulse (0.5 before and after bath applications of peptide-free solution for 10 min. (B) same Fl neuron, resting membrane application is 10 min.
DISCUSSION
The present results clearly demonstrate that the tetrapeptide APGWamide and the dipeptide GWamide are qualitatively and quantitatively similar in their actions on Helix aspersa central neurons. This is true for both their presynaptic and postsynaptic actions. In addition the postsynaptic actions vary in their intensity, depending on the neuron under study. Two identified neurons were used, Fl which is excitated by acetylcholine and where q APGWamide q GWamide
L
0.1
CIM 0.5
PM
1 PM
Wash
Fig. 3. A histogram to show the dose-response relation of APGWamide and GWamide on the amplitude of evoke-IPSPs induced by stimulating the right pailial nerve (0.5 msec, 45 V) on Fl neurons. In normal saline, the amplitude of evoked-IPSPs was as control (loo%), (0) presents the evoked-IPSPs induced after incubation of APGWamide (0.1. 0.5. 1.0 uM) for 2 min. (@) shows in presence of GWamide (0. i, 0.‘5, I.OPM). Wash means that the amplitudes of evoked-IPSPs were induced after washing with peptide-free saline for 10 min. n = 3-5, 5 SE mean.
M. L. CHEN and R. J. WALKER
512
Wamide (Kobayashi and Muneoka, 1990). However, we observed that APGWamide and GWamide have the same potency either on the presynaptic or on the postsynaptic sites in Helix neurons. We suggest that the amino acid sequence, Gly-Trp, might be essential for their neurobiological activity. The relationship between APGWamide and RPCH and also with AKH (adipokinetic hormone of insects) is of interest. The sequence of RPCH, that is, pQLDFSPGWamide, shares the last 3 C-terminal amino acids
APGW-amide and GWamide have very weak postsynaptic actions, and F2 where 500 nM of either peptide has a threshold inhibitory effect while ACh is excitatory but through an increase in chloride permeability. The actions of four peptides, APGWamide, FAPGWamide, RPCH (red pigment concentrating hormone of crustacea) and PGWamide have been tested for their presynaptic actions on a range of molluscan tissues and the order of potency was APGWamide z FAPGWamide > RPCH > PG-
Normal saline
A
1. Control
’ dopamine
2. APGWamide
3.
GWamide
( 100 mM, 40 pai,
200 ms
,
1
1 NM,
2 min
B 20 mM Mg, 0.5 ml4 Ca saline 1. Control
’ dopamino
( 100 mM, 40 pal,
200 me
)
2. APGWamSde 1 PM, 2 min
3. GWamide 1 ILM, 2 min
dopmine
Fig. 4. Effects of APGWamide and GWamide on the evoked-IPSPs and dopamine-induced responses of Fl neuron. Dots indicate that the right pallial nerve was stimulated by a square pulse (0.5 msec, 4 V) and an IPSP was evoked. Arrows indicate the pressure injection of dopamine (100 mM, 40 psi, 200 ms) which induced a hyperpolarizing response. (A) Is in the normal saline and shows (I) control, (2) in presence of APGWamide (I FM, 2 min), (3) GWamide (1 MM), 2min), and (4) washing by peptide-free saline for 15min. (B) Is in high Mg2+, low Ca*+ saline (20 mM Mg2+, 0.5 mM Ca2+) and shows (1) control, (2) APGWamide (1 PM, 2 min) and (3) GWamide (1 PM, 2 min). All the responses were recorded from the same FI neuron, resting membrane potential about -50 mV. The interval of each experiment is 10 min.
Effect of APGW-amide and GW-amide on snail neurons Normal
saline
t ACh
B
NII free saline
-J-L-t ACh
t APGWamide
0.1 U
t
APGWamide
0.5 @I
4
APGWamide
1 MM
t APGWamide
513
5 vl4
7--t ACh
t Wash
t
t
t
t
GWamide
0.1 @4
t Wash
t
GWamide
0.5 !JH
t
4
GWamide
1 PM
t
4 GWamide
5 KM
t
Fig. 5. Effects of APGWamide and GWamide on F2 neuron. (A) Is in normal saline and a depolarized response induced by pressure injection of ACh (1 mM, 40 psi, 10 msec). (B) Shows that following perfusion with Na+ free saline, ACh could induce either a depolarization or hyperpolarization depending on the membrane potential. Note that the reversal potential of ACh-induced response in Nat free saline is about -35/-40mV. (C) and (D) Show the responses in normal saline, following perfusion with different concentrations of APGWamide and GWamide. All the responses were recorded from the same F2 neuron, resting membrane potential about -50 mV. The interval of drug application is 10 min.
while AKH with the sequence, pQLDFTPDWGTamide, shares the first 3 N-terminal amino acids. However, at the C-terminal only the penultimate G is common to the two peptides. It would therefore be of interest to test the dipeptide, GTamide for activity. It would likewise be interesting to examine the actions of both RPCH and AKH on APGWamide sensitive neurons of Helix. In various molluscan muscles, APGWamide modifies the presynaptic release of acetylcholine and S-HT (Kuroki et al., 1990; Kobayashi and Muneoka, 1990). In our study using Helix neurons, we have also shown that APGWamide and GWamide modified the presynaptic release of dopamine. It is therefore likely that APGWamide and GWamide modulate presynaptically the release of a wide range of transmitters in molluscs, possibly including peptide transmitters such as FMRFamide (Walker, 1992). The presynaptic actions of APGWamide and GWamide on dopamine release onto Fl neuron are dose dependent and reversible. The threshold concentration for a presynaptic effect is around 500 nM, with 1 PM producing a very significant reduction in the dopamine IPSP. At concentrations which induced this presynaptic effect, there is no effect on the response to postsynaptic dopamine. Since APGWamide is present in gastropod nerve tissue there will be APGWamide-containing neurons which are likely to synapse presynaptically onto dopamine-containing neurons, (Fig. 9). The postsynaptic inhibitory action of APGWamide and GWamide on F2 neurons would appear to be pure potassium events, with a reversal potential of around -85 mV. This value is reasonably close to
that of -74 mV, obtained by Kostyuk (1968). This inhibitory effect is dependent on external potassium concentration and in the presence of high external potassium, the response is completely abolished. As shown in Fig. 7 there is a good correlation between the size of the hyperpolarization induced by APGWamide and the external potassium concentration. Neither tetraethylammonium nor 4-aminopyridine were able to completely block the APGWamide and GWamide inhibition while a combination of both, each at a lower concentration than when applied alone, almost completely blocked the inhibition. However, neither La2+ nor Co’+ block the inhibition induced by APGWamide and GWamide. This would suggest that both TEA-sensitive and 4-AP-sensitive potassium channels are activated by APGWamide and GWamide. This profile is similar to the I, channel which activates slowly with small depolarization. Recently, it has been shown that APGWamide also inhibits central neurons of Achatina (Liu et al., 1991). This action of APGWamide is also through activation of potassium channels. These authors suggest APGWamide is a postsynaptic inhibitory transmitter in Achatina while FMRFamide and Ser2MIP which are also inhibitory on Achatina neurons, may simply mimic the action of APGWamide. Interestingly FMRFamide has not been shown to occur in Achatina but it would be very surprising if this peptide were in fact absent (Walker, 1992). On Helix Fl neurons, FMRFamide has a potent inhibitory action (Boyd and Walker, 1985) while on F2 FMRFamide has a excitatory action. It is obvious that APGWamide and FMRFamide do not act on the same type of receptors.
514
M. L. CHENand R. J. WALKER
B [K’],
:
4 mM
30
mV
-I 20
s
4
Fig. 6. Effects of changing the external potassium concentration on the membrane hyperpolarization induced by APGWamide. The recordings were from the same F2 neuron and the interval of peptide application was IOmin. A, B, C, D, and E show responses following incubation with K+-free, 4,8,12,16mM of K+ saline for 10 min. The amplitude of membrane hyperpolarizations induced by APGWamide (5 PM) were gradually reduced with increased external K’ concentration. CONCLUSION
1. APGWamide and GWamide, 0.5-S.OpM, reversibly inhibited the amplitude of evoked-IPSPs which were dopaminergic and due to an increase in potassium conductance, but had no direct effect on membrane potential, ceil firing rate, or dopamineinduced responses. These results indicate that the actions of APGWamide and GWamide in F 1 neurons are at presynaptic sites. 2. At concentrations between 0.5 and lOpM, both APGWamjde and GWamide had direct postsynaptic effects on F2 neurons. They inhibited the spike activity and hyperpolarized the membrane potential of F2 neurons in a dose-dependent manner. 3. In K+ free solution, the inhibitory effects of APGWamide and GWamide were potentiated, while they were gradually reduced by increasing external K+ concentration. Applications of potassium channel blockers, either tetraethylammonium (TEA, 10mM) or 4-aminopyridine (4-AP, 500 PM), only partially prevented the inhibitory effects of APGWamide and GWamide on F2 neurons, Combination of TEA (5 mM) and 4-AP (250 PM) could prevent the inhibition induced by APGWamide and
GWamide. This evidence indicates that the actions of APGWamide and GWamide are due to an increase in potassium conductance on F2 neurons and that
Fig. 7. Effects of APGWamide and GWamide in different concentration of K+ saline on F2 neurons. Abscissa, relative [K’], in log scale. Ordinate presents the percent of control of membrane hyperpolarization induced by bath application of 5pM peptides (0: APGWamide. 0: GWamide; n = 4, F: SE mean). The amplitude of membrane hyperpolarization induced in normal saline, [K+], = 4 mM, was taken as control (I~%).
Effect of APGW-amide
A
Nom1
C
10
+ Wash
saline
mM TEA
4 APGWamide
5 pM*
p_M 4-Al?
saline
500
+APGWamideS&
D
5 mM TEA
+
j APGWamide E
Normal
on snailneurons
saline
~APGWamideSp~
B
and GW-amide
GWamide
250
pM
4-AP
5 @4
saline
5 I_IMj
+ GWamide
5 m
+
saline
4 AF'GWamide
5 KM
t
Fig. 8. Actions of K+ channel blockers on the effects of APGWamide and GWamide on F2 neuron. All the recordings were from the same neuron. (A) Bath application of APGWamide and GWamide (5 PM), respectively, in normal saline. (B) In presence of 10 mM TEA saline for 10 min. (C) 500 PM 4-AP saline for 10 min. (D) Combination of 5 mM TEA and 250 PM 4-AP saline for 10 min. (E) Washing with normal saline by 20 min. The resting membrane potential of F2 neuron is about - 50 mV. The interval of each experiment is 20min. Between B and C and between C and D, the preparation was washed in normal saline and control peptide responses obtained.
both transient K channel (I,) and delay K channel (IK) were affected by these peptides. In this study, we conclude that APGWamide and GWamide can exert both presynaptic and postsynaptic effects on central neurons of Helix aspersa depending on the type of neuron. The postsynaptic
Fig. 9. Diagram to summarize possible connections between an APGWamide (APGW) containing neuron and follower cells. The APGW neuron releases peptide which can act presynaptically, for example, at the terminals of a dopamine-containing cell, to modify the release of dopamine onto a follower cell, such as Fl neuron. The peptide can also be released directly onto the postsynaptic membrane of a follower cell, for example, F2, to modify cell activity.
action is associated with an increase in potassium permeability, involving both I, and I, channels. Both peptides have similar potencies on Helix aspersa neurons. REFERENCES Bokisch A. J. and Walker R. J. (1986) The ionic mechanism associated with the action of putative transmitters on identified neurons of the snail, Helix aspersa. Comp. Biochem. Physiol. 84C, 231-241. Boyd P. J. and Walker R. J. (1985) Actions of the molluscan neuropeptide FMRFamide on .neurons in the subesophageal ganglia of the snail, Helix uspersu. Comp. Biochem. Physiol. NC, 379-386. Fernlund P. (1974) Structure of the red-pigment-concentrating hormone of the shrimp, Pundalus borealis. Biochem. Biophys. Acta. 371, 304-311. Greenberg M. J., Rao K. R., Lehman H. K., Price D. A. and Doble K. E. (1985) Cross-phyletic bioactivity of arthropod neurohormones and molluscan ganglion extracts: evidence of an extended peptide family. J. exp. 2001. 233, 3377346. Kerkut G. A., Horn N. and Walker R. J. (1969) Longlasting synaptic inhibition and its transmitter in the snail Helix aspersa. Comp. Biochem. Physiol. 30, 1061-1074. Kerkut G. A., Lambert J. D. C., Gayton R. J., Loker J. E. and Walker R. J. (1975) Mapping of nerve cells in the suboesophageal ganglia of Helix aspersa. Comp. Biochem. Physiol. SUA, l-25.
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Kerkut G. A. and Meech R. W. (1966) The internal chloride concentration of H and D cells in the snail brain. Comp. Biochem. Physiol. 19, 819-832.
Kobayashi M. and Muneoka Y. (1990) Structure and action of molluscan neuropeptides. Zool. Sci. 7, 801-814. Kostyuk P. G. (1968) Ionic background of activity in giant neurones of molluscs. In Neurobiology of Invertebrates (Edited by J. Salanki), pp. 145-167. Plenum Press, New York. Kuroki Y., Kanda T., Kubota I., Fujisawa Y., Ikeda T., Miura A., Minamitake Y. and Muneoka Y. (1990) A molluscan neuropeptide related to the crustacean hormone, RPCH. Biochem. Biophys Res. Comm. 167, 213-279. Liu G. J., Santos D. E., Takeuchi H., Kamatani Y., Minakata H., Nomoto K., Kubota I., Ikeda T. and
WALKER
Muneoka Y. (1991) APGWamide as an inhibitory neurotransmitter of Achatina filica ferussac. Biochem. Biophys. Res. Comm. 177, 27-33.
Stone J. S., Mordue W., Batley K. E. and Morris H. R. (1976) Structure of locust adipokinetic hormone, a neurohormone that regulates lipid utilisation during flight. Nature 263, 207-211.
Yanagawa M., Fujiwara M., Takabatake I., Muneoka Y. and Kobayashi M. Potentiating effects of some invertebrate neuropeptides on twitch contraction of the radula muscles of mollusc, Rapana thomasiana. Camp. Biochem. Physiol. 9QC, 13-77.
Walker R. J. (1992) Neuroactive peptides with an RFamide or Famide carboxyl terminal: a mini review. Camp. B&hem. Physiol. (in press).
0306-4492/92 $5.00 + 0.00 Q 1992 Pergamon Press Ltd
Printed in Great Britain
ACTIONS OF APGW-AMIDE AND GW-AMIDE ON IDENTIFIED CENTRAL NEURONS OF THE SNAIL, HELIX ASPERSA M. L. CHEN and R. J. WALKER Department
of Physiology and Pharmacology, University of Southampton, Bassett Crescent East, Southampton, SO9 3TU, UK (Tel.: (0703) 594348 (Fax: (0703) 594319) (Received 17 October 1991)
Abstract-l. Actions of APGWamide and GWamide have been examined on identified central neurons of the snail, Helix aspersa. 2. In Fl neurons, both APGWamide and GWamide, 0.5-5 PM, reversibly inhibited the amplitude of evoked-IPSPs which were dopaminergic, but had no direct effect on membrane potential, cell firing rate, or dopamine-induced responses. These results indicate that the actions of APGWamide and GWamide in Fl neurons are at presynaptic sites. 3. At concentrations between 0.5 and lOpM, APGWamide and GWamide had direct postsynaptic effects on F2 neurons. They inhibited the spike activity and hyperpolarized the membrane potential of F2 neurons in a dose-dependent manner with a reversal potential around -85 mV which is close to E, 4. In K+ free solution, the inhibitory effects of APGWamide and GWamide were potentiated, while they were reduced by increasing external K+ concentration. Either tetraethylammonium (10mM) or 4-aminopyridine (500 PM) only partially prevented the inhibition induced by APGWamide and GWamide on F2 neurons. Combination of TEA (5 mM) and 4-AP (250 PM) could abolish this inhibition. However, neither 1 mM La*+ nor 10 mM Co2+ could prevent the inhibitory action of APGWamide and GWamide. This evidence indicates that the postsynaptic inhibition of APGWamide and GWamide on F2 neurons is due to an increase in K+ conductance and that both transient K channel (IA) and delay K+ channel (IK) were affected by these peptides. 4. APGWamide and GWamide exert both presynaptic and postsynaptic effects of Helix neurons,
depending on the neuron under study. They are qualitatively and quantitatively similar in their presynaptic or postsynaptic actions. We suggest that the amino acid sequence, Gly-Trp, might be essential to their neurobiological activity.
INTRODUCTION
A number of arthropod neuropeptides have been identified since 1974. The first one was the crustacean red-pigment-concentrating hormone (RPCH), isolated from shrimps (Femlund, 1974). Adipokinetic hormone (AKH) was isolated from locusts and its primary structure is closely related to RPCH (Stone et al., 1976). RPCH and AKH have biological actions on arthropod and molluscan tissues. The locust AKH and the crustacean RPCH have excitatory effects on the heart of the clam Mercenaria mercenuria (Greenberg et al., 1985). RPCH also shows modulatory actions on contractions of molluscan muscles (Yanagawa et al., 1988). Recently, a tetrapeptide, APGWamide, has been isolated from the ganglia of the prosobranch mollusc Fusinus ferrugineus and the ganglia of African giant snail, Achatina fulica Ferussac (Kuroki et al., 1990; Liu et al., 1991). The primary structure of APGWamide is related to the C-terminal tetrapeptide fragment of RPCH (Kuroki er al., 1990). This peptide was found to have a presynaptic modulatory action on contractions in various molluscan muscles (Kobayashi and Muneoka, 1990) and a postsynaptic inhibitory action on Achatina neurons, due to an increase in K+ conductance (Liu et al., 1991). However, the type of K+ channel involved in the neuronal modulation is unknown. The aim of this study is to elucidate the mode of action of APGW-
amide on the central neurons of Helix aspersa and to compare its actions with another synthetic peptide GWamide (H-Gly-Trp-NH,), as part of a structure-activity study. METHODS AND
MATERIALS
Experiments were performed on identified neurons of suboesophageal ganglia of the snail, Helix ospersa (Kerkut et al., 1975). The isolated ganglia were pinned on a small Sylgard-coated chamber with 1 ml volume and were immersed in normal saline which contained the following components (mM): NaCl 100, KC1 4, CaCl, 7, MgCl, 5, Tris base 5, final pH 7.5. Intracellular recordings were made using glass microelectrodes filled with 2 molar potassium acetate and with a resistance of 5-10 MR. The electrode was connected by a Ag-AgCl wire to a Dagan single electrode voltage-clamp system in the current clamp mode. Signals were displayed on a Tektronix 5111A oscillosope and permanent traces made using a Hewlett Packard 7402A chart recorder. In some experiments, the right pallial nerve trunk was sucked into a plastic suction electrode and stimulus pulses were produced by a Grass D9 stimulator. The duration of the stimulus pulse was between 0.5 and 2 msec, but the intensity of stimulation was varied. Application of chemicals was either by bath perfusion or by pressure ejection through a picospritzer II pressure ejector. The inner diameter of the tip of ejection electrodes is 2-3 pm. During the experiments, saline was continuously perfused over the preparation with perfusion rate 5% ml per min. In Na-free saline, sodium was replaced by equal 509
M. L. CHEN and R.J.
510
molar Tris base,CaClz 2 mM, MgC1, 10 mM, KC1 4 mM, using 12 molar HCI to adjust pH to 7.5. APGWamide: a gift from Professor Y. Muneoka (Hiroshima, University, Japan) GWamide: purchased from Bachem Feinchmikalien AG/Switzerland. RESULTS
In Fl neurons, 5 PM acetylcholine (ACh) depolarized the membrane and increased the cell firing rate. The ACh-induced response was sodium dependent, since it was abolished when Na’ was replaced by equal molar Tris in normal saline (Fig. IA). Bath application of APGWamide and GWamide (0.5, 1 PM) had no direct effect either on the membrane potential or on the cell firing rate of Fl neurons. However, at IOpM, both peptides slightly decreased cell firing rate (Fig. lB,C). An inhibitory postsynaptic potential (IPSP) could be evoked in Fl neurons when the right pallial nerve trunk was stimulated by a suction electrode (Fig. 2A. control). These evoked-IPSPs are dopaminergic and due to an increase in potassium conductance (Kerkut et al., 1969). When the amplitude of evoked-IPSPs was taken as 100% in normal saline, application of APGWamide 0.1, 0.5 and I PM for 2 min, reduced the amplitude of evoked-IPSP to 95.0 f 9.8%, 62.7 * 7.2%, and 38.3 -t_ 11.2% respectively of control (n = 4, f SEM) (Fig. 2A, Fig. 3). After washing with peptide-free saline for IOmin, the amplitude of evoked-IPSPs could be partially restored to 88.5 k 3.3% of control (n = 4, k SEM). GWamide also showed reversible inhibition on the evokedIPSPs in the same Fl neuron (Fig. 2B). In the presence of GWamide 0.1, 0.5 and 1 PM for 2 min, the amplitude of evoked-IPSPs was decreased to
Normal
Na
Saline
+AKWamide
0.5 JIM
tAPGWamide 1 p14
+AF%Wamide 10 J.IM
WALKEK
77.6 + 8.5%, 60.3 _t 9.7%, and 37.3 + 2.5% respectively of control (n = 4-5, + SEM). Following washing, the IPSPs returned to 90.7 _+9.3% of control (n = 4, + SEM, Fig. 3). A hyperpolarization could be produced by pressure ejection of dopamine (100 mM, 40 psi, 200 msec; Fig. 4A-1). In the presence of APGWamide and GWamide (I PM, 2 min), the amplitude of evokedIPSPs was reduced but the dopamine-induced responses were still present (Fig. 4A-2, 3). After washing with peptide-free saline, the amplitude of evoked-IPSPs was restored (Fig. 4A-4). In the presence of peptides, some synaptic potentials appeared in the period of dopamine-induced responses, so the actions of peptides in high Mg2+, low Ca2+ saline (20mMMgZ+,0.5mmCa 2+, Fig. 4B) were examined. In the presence of this saline, synaptic activity of presynaptic neurons could be abolished and so the direct effect of peptides on the identified neurons could be examined under these conditions. The dopamine-induced responses were not affected by either APGWamide or GWamide (I PM, 2 min, Fig. 4B2, 3). APGWamide and GWamide, at concentrations between 0.5 and 1 PM, showed dose-dependent inhibition on evoked-IPSPs in Fl neurons, but they had no direct effect on membrane potentials, cell firing rate or dopamine-induced responses. These results indicated that the actions of APGWamide and GWamide are on presynaptic sites. In contrast to Fl neurons, APGWamide and GWamide showed direct inhibitory effects on F2 neurons. In normal saline, pressure ejections of ACh (1 mM, 40 psi, 10 msec) could produce a depolarization (Fig. 5A). However, in the presence of Na+ free saline, ACh still evoked either a depolarization or hyperpolarization in F2 neurons, depending on the
saline
free
+Wash
t
t GWamide 0.5 uM
(GWamide 1 p4
twash
+
4 GWamide 10 ,uM
Fig. 1. Effects of APGWamide and GWamide on the spontaneous spike activity of Fl neuron in the suboesophageal ganglia of Hdiu asprrscr. (A) In normal saline, bath application of 5 PM ACh depolarized the neuron and increased the spike activity, whereas in the Na+ free solution, the ACh-induced response disappeared. Uparrows (t) indjcate when hrugs were bath applied and downarrows (1) show when drugs were washed bvi normal saline. (B) _ / Shows responses to 0.5, 1.O. 10 PM APGWamide. (C) Shows responses to 0.5, 1.0, 10 pM GWamide. All the intracellular recordings are from the same Fl neuron, resting membrane potential about -6OmV. The interval of peptide application is 1Omin.
Effect of
A
B
Control
Control
APGW-amide and GW-amide on snail neurons
APGWamide 0.1 t.LM
0.5
1.0
uM
Wash
n&f
aamide 0.1
Wash
j.l&l
30 mv --I 20 s Fig. 2. Effects of APGWamide and GWamide on the amplitude indicate that an IPSP could be evoked when the right pallial nerve ms, 4 V) through a plastic suction electrode. (A) Shows responses APGWamide, 0.1, 0.5, 1.0 FM, for 2 min, and then washing with Shows the actions of GWamide on the evoked-IPSPs from the potential about - 50 mV. The interval of peptide
membrane potential levels (Fig. 5B). The AChinduced responses on F2 neurons are chloride dependent and have a reversal potential of -35/-40mV (Kerkut and Meech, 1966). Both APGWamide and GWamide (0.5, 1, 5 PM) blocked cell firing and hyperpolarized the membrane potential in a dose-dependent manner of F2 neuron, (Fig. 5 CD). The reversal potential for this inhibitory event is around - 85 mV, close to E, in Helix neurons (Kostyuk, 1968; Bokisch and Walker 1986). The extent of membrane hyperpolarization induced by APGWamide (5 KM) on F2 neuron was affected by changing the external K+ concentration. In normal saline, the external potassium concentration is 4 mM and 5 ~1M APGWamide could produce hyperpolarizing responses of 16.7 + 1.OmV (n = 3 + SEM; Fig. 6B). In K+-free saline, the hyperpolarization induced by peptide was increased to 23.3 &-1.0 mV (n = 3, +_SEM; Fig. 6A). The amplitude of the hyperpolarization was reduced by increasing external K+ concentration (Fig. 6 C,D,E). The relationship between membrane hyperpolarization induced by APGWamide and GWamide (5 PM) and external K+ concentration is shown in Fig. 7. Fig. 8A shows that inhibition was induced by both 5,~cM of APGWamide and GWamide in normal saline. In the presence of either 10 mM tetraethyl ammonium (TEA) or 500 p M 4-aminopyridine (4AP), the inhibitory actions could be partially blocked by either K+ channel blocker (Fig. 8B,C). However, a combination of 5 mM TEA and 250pM 4-AP abolished the inhibition induced by peptides in F2 neurons (Fig. 8D). After washing with normal saline, the inhibition induced by either peptide could be restored, Fig. 8E shows the recovery to APGWamide (5pM). Furthermore, we also found that neither 10 mM Co’+ nor 1 mM La’+ could block the inhibition induced by both APGWamide and GWamide (5 p M) on F2 neurons.
of evoked-IPSPs of Fl neuron. Dots was stimulated by a square pulse (0.5 before and after bath applications of peptide-free solution for 10 min. (B) same Fl neuron, resting membrane application is 10 min.
DISCUSSION
The present results clearly demonstrate that the tetrapeptide APGWamide and the dipeptide GWamide are qualitatively and quantitatively similar in their actions on Helix aspersa central neurons. This is true for both their presynaptic and postsynaptic actions. In addition the postsynaptic actions vary in their intensity, depending on the neuron under study. Two identified neurons were used, Fl which is excitated by acetylcholine and where q APGWamide q GWamide
L
0.1
CIM 0.5
PM
1 PM
Wash
Fig. 3. A histogram to show the dose-response relation of APGWamide and GWamide on the amplitude of evoke-IPSPs induced by stimulating the right pailial nerve (0.5 msec, 45 V) on Fl neurons. In normal saline, the amplitude of evoked-IPSPs was as control (loo%), (0) presents the evoked-IPSPs induced after incubation of APGWamide (0.1. 0.5. 1.0 uM) for 2 min. (@) shows in presence of GWamide (0. i, 0.‘5, I.OPM). Wash means that the amplitudes of evoked-IPSPs were induced after washing with peptide-free saline for 10 min. n = 3-5, 5 SE mean.
M. L. CHEN and R. J. WALKER
512
Wamide (Kobayashi and Muneoka, 1990). However, we observed that APGWamide and GWamide have the same potency either on the presynaptic or on the postsynaptic sites in Helix neurons. We suggest that the amino acid sequence, Gly-Trp, might be essential for their neurobiological activity. The relationship between APGWamide and RPCH and also with AKH (adipokinetic hormone of insects) is of interest. The sequence of RPCH, that is, pQLDFSPGWamide, shares the last 3 C-terminal amino acids
APGW-amide and GWamide have very weak postsynaptic actions, and F2 where 500 nM of either peptide has a threshold inhibitory effect while ACh is excitatory but through an increase in chloride permeability. The actions of four peptides, APGWamide, FAPGWamide, RPCH (red pigment concentrating hormone of crustacea) and PGWamide have been tested for their presynaptic actions on a range of molluscan tissues and the order of potency was APGWamide z FAPGWamide > RPCH > PG-
Normal saline
A
1. Control
’ dopamine
2. APGWamide
3.
GWamide
( 100 mM, 40 pai,
200 ms
,
1
1 NM,
2 min
B 20 mM Mg, 0.5 ml4 Ca saline 1. Control
’ dopamino
( 100 mM, 40 pal,
200 me
)
2. APGWamSde 1 PM, 2 min
3. GWamide 1 ILM, 2 min
dopmine
Fig. 4. Effects of APGWamide and GWamide on the evoked-IPSPs and dopamine-induced responses of Fl neuron. Dots indicate that the right pallial nerve was stimulated by a square pulse (0.5 msec, 4 V) and an IPSP was evoked. Arrows indicate the pressure injection of dopamine (100 mM, 40 psi, 200 ms) which induced a hyperpolarizing response. (A) Is in the normal saline and shows (I) control, (2) in presence of APGWamide (I FM, 2 min), (3) GWamide (1 MM), 2min), and (4) washing by peptide-free saline for 15min. (B) Is in high Mg2+, low Ca*+ saline (20 mM Mg2+, 0.5 mM Ca2+) and shows (1) control, (2) APGWamide (1 PM, 2 min) and (3) GWamide (1 PM, 2 min). All the responses were recorded from the same FI neuron, resting membrane potential about -50 mV. The interval of each experiment is 10 min.
Effect of APGW-amide and GW-amide on snail neurons Normal
saline
t ACh
B
NII free saline
-J-L-t ACh
t APGWamide
0.1 U
t
APGWamide
0.5 @I
4
APGWamide
1 MM
t APGWamide
513
5 vl4
7--t ACh
t Wash
t
t
t
t
GWamide
0.1 @4
t Wash
t
GWamide
0.5 !JH
t
4
GWamide
1 PM
t
4 GWamide
5 KM
t
Fig. 5. Effects of APGWamide and GWamide on F2 neuron. (A) Is in normal saline and a depolarized response induced by pressure injection of ACh (1 mM, 40 psi, 10 msec). (B) Shows that following perfusion with Na+ free saline, ACh could induce either a depolarization or hyperpolarization depending on the membrane potential. Note that the reversal potential of ACh-induced response in Nat free saline is about -35/-40mV. (C) and (D) Show the responses in normal saline, following perfusion with different concentrations of APGWamide and GWamide. All the responses were recorded from the same F2 neuron, resting membrane potential about -50 mV. The interval of drug application is 10 min.
while AKH with the sequence, pQLDFTPDWGTamide, shares the first 3 N-terminal amino acids. However, at the C-terminal only the penultimate G is common to the two peptides. It would therefore be of interest to test the dipeptide, GTamide for activity. It would likewise be interesting to examine the actions of both RPCH and AKH on APGWamide sensitive neurons of Helix. In various molluscan muscles, APGWamide modifies the presynaptic release of acetylcholine and S-HT (Kuroki et al., 1990; Kobayashi and Muneoka, 1990). In our study using Helix neurons, we have also shown that APGWamide and GWamide modified the presynaptic release of dopamine. It is therefore likely that APGWamide and GWamide modulate presynaptically the release of a wide range of transmitters in molluscs, possibly including peptide transmitters such as FMRFamide (Walker, 1992). The presynaptic actions of APGWamide and GWamide on dopamine release onto Fl neuron are dose dependent and reversible. The threshold concentration for a presynaptic effect is around 500 nM, with 1 PM producing a very significant reduction in the dopamine IPSP. At concentrations which induced this presynaptic effect, there is no effect on the response to postsynaptic dopamine. Since APGWamide is present in gastropod nerve tissue there will be APGWamide-containing neurons which are likely to synapse presynaptically onto dopamine-containing neurons, (Fig. 9). The postsynaptic inhibitory action of APGWamide and GWamide on F2 neurons would appear to be pure potassium events, with a reversal potential of around -85 mV. This value is reasonably close to
that of -74 mV, obtained by Kostyuk (1968). This inhibitory effect is dependent on external potassium concentration and in the presence of high external potassium, the response is completely abolished. As shown in Fig. 7 there is a good correlation between the size of the hyperpolarization induced by APGWamide and the external potassium concentration. Neither tetraethylammonium nor 4-aminopyridine were able to completely block the APGWamide and GWamide inhibition while a combination of both, each at a lower concentration than when applied alone, almost completely blocked the inhibition. However, neither La2+ nor Co’+ block the inhibition induced by APGWamide and GWamide. This would suggest that both TEA-sensitive and 4-AP-sensitive potassium channels are activated by APGWamide and GWamide. This profile is similar to the I, channel which activates slowly with small depolarization. Recently, it has been shown that APGWamide also inhibits central neurons of Achatina (Liu et al., 1991). This action of APGWamide is also through activation of potassium channels. These authors suggest APGWamide is a postsynaptic inhibitory transmitter in Achatina while FMRFamide and Ser2MIP which are also inhibitory on Achatina neurons, may simply mimic the action of APGWamide. Interestingly FMRFamide has not been shown to occur in Achatina but it would be very surprising if this peptide were in fact absent (Walker, 1992). On Helix Fl neurons, FMRFamide has a potent inhibitory action (Boyd and Walker, 1985) while on F2 FMRFamide has a excitatory action. It is obvious that APGWamide and FMRFamide do not act on the same type of receptors.
514
M. L. CHENand R. J. WALKER
B [K’],
:
4 mM
30
mV
-I 20
s
4
Fig. 6. Effects of changing the external potassium concentration on the membrane hyperpolarization induced by APGWamide. The recordings were from the same F2 neuron and the interval of peptide application was IOmin. A, B, C, D, and E show responses following incubation with K+-free, 4,8,12,16mM of K+ saline for 10 min. The amplitude of membrane hyperpolarizations induced by APGWamide (5 PM) were gradually reduced with increased external K’ concentration. CONCLUSION
1. APGWamide and GWamide, 0.5-S.OpM, reversibly inhibited the amplitude of evoked-IPSPs which were dopaminergic and due to an increase in potassium conductance, but had no direct effect on membrane potential, ceil firing rate, or dopamineinduced responses. These results indicate that the actions of APGWamide and GWamide in F 1 neurons are at presynaptic sites. 2. At concentrations between 0.5 and lOpM, both APGWamjde and GWamide had direct postsynaptic effects on F2 neurons. They inhibited the spike activity and hyperpolarized the membrane potential of F2 neurons in a dose-dependent manner. 3. In K+ free solution, the inhibitory effects of APGWamide and GWamide were potentiated, while they were gradually reduced by increasing external K+ concentration. Applications of potassium channel blockers, either tetraethylammonium (TEA, 10mM) or 4-aminopyridine (4-AP, 500 PM), only partially prevented the inhibitory effects of APGWamide and GWamide on F2 neurons, Combination of TEA (5 mM) and 4-AP (250 PM) could prevent the inhibition induced by APGWamide and
GWamide. This evidence indicates that the actions of APGWamide and GWamide are due to an increase in potassium conductance on F2 neurons and that
Fig. 7. Effects of APGWamide and GWamide in different concentration of K+ saline on F2 neurons. Abscissa, relative [K’], in log scale. Ordinate presents the percent of control of membrane hyperpolarization induced by bath application of 5pM peptides (0: APGWamide. 0: GWamide; n = 4, F: SE mean). The amplitude of membrane hyperpolarization induced in normal saline, [K+], = 4 mM, was taken as control (I~%).
Effect of APGW-amide
A
Nom1
C
10
+ Wash
saline
mM TEA
4 APGWamide
5 pM*
p_M 4-Al?
saline
500
+APGWamideS&
D
5 mM TEA
+
j APGWamide E
Normal
on snailneurons
saline
~APGWamideSp~
B
and GW-amide
GWamide
250
pM
4-AP
5 @4
saline
5 I_IMj
+ GWamide
5 m
+
saline
4 AF'GWamide
5 KM
t
Fig. 8. Actions of K+ channel blockers on the effects of APGWamide and GWamide on F2 neuron. All the recordings were from the same neuron. (A) Bath application of APGWamide and GWamide (5 PM), respectively, in normal saline. (B) In presence of 10 mM TEA saline for 10 min. (C) 500 PM 4-AP saline for 10 min. (D) Combination of 5 mM TEA and 250 PM 4-AP saline for 10 min. (E) Washing with normal saline by 20 min. The resting membrane potential of F2 neuron is about - 50 mV. The interval of each experiment is 20min. Between B and C and between C and D, the preparation was washed in normal saline and control peptide responses obtained.
both transient K channel (I,) and delay K channel (IK) were affected by these peptides. In this study, we conclude that APGWamide and GWamide can exert both presynaptic and postsynaptic effects on central neurons of Helix aspersa depending on the type of neuron. The postsynaptic
Fig. 9. Diagram to summarize possible connections between an APGWamide (APGW) containing neuron and follower cells. The APGW neuron releases peptide which can act presynaptically, for example, at the terminals of a dopamine-containing cell, to modify the release of dopamine onto a follower cell, such as Fl neuron. The peptide can also be released directly onto the postsynaptic membrane of a follower cell, for example, F2, to modify cell activity.
action is associated with an increase in potassium permeability, involving both I, and I, channels. Both peptides have similar potencies on Helix aspersa neurons. REFERENCES Bokisch A. J. and Walker R. J. (1986) The ionic mechanism associated with the action of putative transmitters on identified neurons of the snail, Helix aspersa. Comp. Biochem. Physiol. 84C, 231-241. Boyd P. J. and Walker R. J. (1985) Actions of the molluscan neuropeptide FMRFamide on .neurons in the subesophageal ganglia of the snail, Helix uspersu. Comp. Biochem. Physiol. NC, 379-386. Fernlund P. (1974) Structure of the red-pigment-concentrating hormone of the shrimp, Pundalus borealis. Biochem. Biophys. Acta. 371, 304-311. Greenberg M. J., Rao K. R., Lehman H. K., Price D. A. and Doble K. E. (1985) Cross-phyletic bioactivity of arthropod neurohormones and molluscan ganglion extracts: evidence of an extended peptide family. J. exp. 2001. 233, 3377346. Kerkut G. A., Horn N. and Walker R. J. (1969) Longlasting synaptic inhibition and its transmitter in the snail Helix aspersa. Comp. Biochem. Physiol. 30, 1061-1074. Kerkut G. A., Lambert J. D. C., Gayton R. J., Loker J. E. and Walker R. J. (1975) Mapping of nerve cells in the suboesophageal ganglia of Helix aspersa. Comp. Biochem. Physiol. SUA, l-25.
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Kerkut G. A. and Meech R. W. (1966) The internal chloride concentration of H and D cells in the snail brain. Comp. Biochem. Physiol. 19, 819-832.
Kobayashi M. and Muneoka Y. (1990) Structure and action of molluscan neuropeptides. Zool. Sci. 7, 801-814. Kostyuk P. G. (1968) Ionic background of activity in giant neurones of molluscs. In Neurobiology of Invertebrates (Edited by J. Salanki), pp. 145-167. Plenum Press, New York. Kuroki Y., Kanda T., Kubota I., Fujisawa Y., Ikeda T., Miura A., Minamitake Y. and Muneoka Y. (1990) A molluscan neuropeptide related to the crustacean hormone, RPCH. Biochem. Biophys Res. Comm. 167, 213-279. Liu G. J., Santos D. E., Takeuchi H., Kamatani Y., Minakata H., Nomoto K., Kubota I., Ikeda T. and
WALKER
Muneoka Y. (1991) APGWamide as an inhibitory neurotransmitter of Achatina filica ferussac. Biochem. Biophys. Res. Comm. 177, 27-33.
Stone J. S., Mordue W., Batley K. E. and Morris H. R. (1976) Structure of locust adipokinetic hormone, a neurohormone that regulates lipid utilisation during flight. Nature 263, 207-211.
Yanagawa M., Fujiwara M., Takabatake I., Muneoka Y. and Kobayashi M. Potentiating effects of some invertebrate neuropeptides on twitch contraction of the radula muscles of mollusc, Rapana thomasiana. Camp. Biochem. Physiol. 9QC, 13-77.
Walker R. J. (1992) Neuroactive peptides with an RFamide or Famide carboxyl terminal: a mini review. Camp. B&hem. Physiol. (in press).
