Comp. Biochem. Physiol., 1975. Vol. 51C, pp. 195 to 203. Pergamon Press. Printed in Great Britain
F U R T H E R STUDIES ON THE ACTION OF VARIOUS 5 - H Y D R O X Y T R Y P T A M I N E AGONISTS A N D A N T A G O N I S T S ON THE RECEPTORS OF N E U R O N E S FROM THE LEECHES, HIRUDO MEDICINALIS A N D HAEMOPIS SANGUISUGA P. A. SMITH AND R. J. WALKER Department of Physiology and Biochemistry, University of Southampton, Southampton SO9 5NH, England (ReceiL,ed 28 January 1975)
Abstract--1. A hypothetical model for the nature of the receptor site involved in the hyperpolarization of leech Retzius cells by 5-hydroxytryptamine (5-HT) is proposed as a result of structure activity studies. 2. The response is best antagonized by the 5-HT M-receptor antagonists atropine and morphine. 3. The current-voltage relationship of the Retzius cells is shown to account for the behaviour of the 5-HT response on hyperpolarization. 4. 5-HT D-receptor antagonists as well as N-methylated and 5-methoxylated tryptamines tend to depolarize Retzius cells, probably by a similar mechanism which may be independent of 5-HT receptors. 5. Annelid 5-HT receptors are compared with those found in molluscs.
INTRODUCTION
THE PRESENCEof 5-hydroxytryptamine (5-HT) within the nervous system of both vertebrates and invertebrates is widely documented (Twarog & Page, 1953; Amins et al., 1954; Welsh & Moorhead, 1960; Welsh, 1968). The possible neurotransmitter role of this monoamine has been most carefully demonstrated in invertebrates in which the responses of single identified neurones may be examined (Gerscbenfeld & Stefani, 1968; Walker et aL, 1972; Gerschenfeld & Paupardin-Tritsch, 1973; Cottrell & Macon, 1974). Most of the studies on the receptors activated by 5-HT have been confined to molluscs. Walker & Woodruff (1972) performed a structure-activity study on neurones excited by 5-HT within the central nervous system of Helix aspersa. Gerscbenfeld (1973) and Gerschenfeld & Paupardin (1973) have defined three serotonin receptors in the central nervous system of Helix. Each receptor specifically increases the membrane permeability to a certain ion or ions and can be blocked by specific antagonists. One of the aims of the present study is to compare the serotonin receptors of identified neurones in the central nervous systems of the leeches Hirudo medicinalis and Haemopis sanguisuga with those in molluscs. Each segmental ganglion of the leech contains a pair of giant neurones (Retzius, 1891). The resting potential of these neurones is hyperpolarized by 5-HT (Kerkut & Walker, 1967), the response being
mediated by an increase in permeability to chloride ions (Walker & Smith, 1973). On hyperpolarization below the calculated equilibrium potential, this response does not reverse. Gerschenfeld & Paupardin (1973) have suggested that such irreversibility may be associated with neurones exhibiting anomalous rectification (Kandel & Tauc, 1966). The current/voltage relationship of the Retzius cells is therefore examined in the present study in an attempt to explain the irreversibility of the 5-HT response. A structure-activity study on the serotonin receptors of these neurones has already been performed (Smith & Walker, 1974). This study is extended in the present paper and a hypothetical model for the receptor site proposed. Certain Nmethylated and 5-methoxylated tryptamines were shown to excite Retzius cells rather than hyperpolarize them. This phenomenon is further investigated in the present study. Most of the experiments described were performed on Hirudo, but occasionally Haemopis had to be substituted when Hirudo was not obtainable. There is apparently some species difference in the sensitivity of the Retzius ceils of these two leeches to certain antagonists and agonists. MATERIALS A N D M E T H O D S
Electrophysiological recordings were made with intracellular glass microelectrodes from the Retzius cells within the central nervous systems of Hirudo medicinalis
195
196
P.A. SMITHAND R. J. WALKER
or Haemopis sanguisuga according to the method of Walker & Smith (1973). The relative potencies of agonists in hyperpolarizing the membrane potential of these neurones were compared by determination of their equipotent molar ratios (e.p.m.r.s.); the ratio of the number of nmol of analogue to 5-HT producing comparable responses (Smith & Walker, 1974). Electrodes for iontophoresis were "fibre filled" with a molar solution of 5-HT bimaleinate brought to pH 4 by the addition of tartaric acid. To examine the properties of various potential antagonists, a standard iontophoretic pulse (500-1000 nA for 5 sec; 2'5-5.0 × l0 s coulombs) of 5-HT bimaleinate was administered and the resulting hyperpolarization noted. No retaining current was required and the indolealkylaminewas ejected as a cation. Three min later a solution of antagonist (I0-~-10-a M) was introduced into the experimental bath. The response to the standard iontophoretic pulse of 5-HT was examined at 3-rain intervals during incubation with the antagonist. If and when blockade was achieved, the antagonist solution was removed by prolonged washing with leech Ringer. A final dose of 5-HT was then administered to assess the reversibility of the antagonism. Two barrels of a triple-barrelled microelectrode filled with M potassium acetate at pH 7 were used to investigate the current/ voltage relationships of Retzius cells. The output of a Grass $44 stimulator was connected via a 100 MO resistor so that a range of hyperpolarizing constant current square wave pulses could be passed through one barrel of the electrode whilst the voltage developed was monitored by the other barrel and displayed on the screen of a Telequipment DMS53A storage oscilloscope triggered from the stimulator. The voltage output from the stimulator was set to deliver a range of pulses from 1 to 10 nA and a permanent record of the voltages developed by these currents obtained with a "Shackman" polaroid camera. The following drugs were used in this study: 6-aminohexanol (Aldrich), 5-methoxy N,N-dimethyltryptamine (Aldrich), 5-methoxygramine (Aldrich), 3(3-Aminopropyl) indole (gift from Dr. M. J. Berridge), atropine sulphate (B.D.H.), 5-hydroxytryptamine creatinine sulphate (B.D.H.), morphine hydrochloride (B.D.H.), bufotenine bioxalate (Sandoz), methylsergide bimaleate (Sandoz), Sandoz MY25 (Sandoz), 2-bromolysergic acid diethylamide (BOL) (Sigma), 5,6-dihydroxytryptamine creatinine sulphate (Sigma), Iproniazid phosphate (Sigma) BW 545 C64 (Tosylate) (gift from Burroughs Wellcome Ltd.), clonidine hydrochloride (gift from Boehringer), Cyproheptadine (Merck, Sharp and Dohme), dibenamine hydrochloride (K and K), 5,7-dihydroxytryptamine creatinine sulphate (Regis), 6-hydroxytryptamine creatinine sulphate (Regis), 7-hydroxytryptamine creatinine sulphate (Regis), Gramine (Emanuel), 3-phenyl-l-propylamine (Emanuel), 5-hydroxytryptamine bimaleinate (Koch-Light), 7-methyltryptamine oxalate (Koch-Light), neostigmine bromide (Koch-Light), phenyldiguanide hydrochloride (Koch-Light), tubocurarine hydrochloride (Koch-Light), mianserin hydrochloride (gift from Organon), O18 Oxalate (gift from Professor E. J. Ariens). RESULTS
Agonists The structures of the agonists used are shown in Fig. 1. 6-Aminohexanol was tested as a 5-HT like
agonist since it resembles the indolealkylamine in that the distance between the terminal hydroxyl groups and amino groups of both compounds are similar but 6-aminohexanol has no aromaticity.
R3
H NH 2
c H O ~
~ NH2
D
~ N H
2
H
H
H2
Fig. I. The structures of some serotonin-like compounds used in this study. (A) Tryptamine derivatives--5-HT: R 1 = O H , R 2 = H , R a = H ; 6-HT: R I = H , R z = O H , R 3 = H; 7-HT: R 1 = H, R 2 = H, Ra = OH; 5,6-diHT: R 1 = O H , R 2 = O H , R a = H ; 5,7-diHT: R 1 = O H , R~ = H, R3 = OH. (B) 3-Phenyl-l-propylamine. ((2) 6Aminohexanol. (D) Gramine. (E) 3(3-Aminopropyl) indole. This compound was inactive on the Retzius cells of the central nervous system of Hirudo in three preparations tested. 3-Phenyl-l-propylamine resembles tryptamine except the 5-membered nitrogen containing ring is omitted. The nitrogen of this ring has been shown to be unimportant in the interaction of 5-HT at the present receptor (Smith & Walker, 1974). In seven experiments this compound failed to exhibit 5-HT like activity in Hirudo. Instead of hyperpolarization dose-dependent depolarizations were observed. 7-Hydroxytryptamine (7-HT), 5,6dihydroxytryptamine (5,6-diHT) and 5,7-dihydroxytryptamine (5,7-diHT) were tested as agonists on Haemopis and exhibited e.p.m.r.'s of 6.0, 4.9 and 1.6 respectively (mean of six experiments). The high potency of these analogues may be explained in terms of the model receptor proposed for Hirudo (Figs. 5 and 6) although another hydroxylated tryptamine (6-HT) was found to exhibit a higher potency in Haemopis than Hirudo. The e.p.m.r, of this compound in Haemopis was 81 for six experiments which compares with a value of 296 in ttirudo (Smith & Walker, 1974). The general high potencies observed for all hydroxylated tryptamines may indicate that the 5-HT receptors in this species are less specific than those in Hirudo.
Studies on leech 5-HT receptors
197
A
10 mv'r/
8 0 0 n A 5HT. i 5 sec.
B
800nA 5HT.
^ W
A f t e r 6min. in 10-4M
Morphine.
C [
,
:
;
;
:
. . . .
, ~'
'
, r L~
~,
t
t: ~,,
~ ~;:
~ ~
'
;
,
Ir~il
~
,
,
~
F
I
800nA 5HT.
!
W
A f t e r 3rain.
D
:::;;
/
;
,' I
,
i'/ I/ I////!/
,i dr f /il/t fIil
^
800nA 3min
5HT
later.
E ,
. . . . . . . .
,, . . . .
I,
3 min
~ ,,
'
!
later
r, , . ,
....
.... , ,
i ,i i, i
~
....
....
,i,, .... 'tj
,,:''
~,
~,l
~
,
~,
t
i:
,i
i,
I / / /{/{ / /
/
I
! ~
800nA
I | t
5HT.
Fig. 2. The effect of 10-4 M morphine on the response of Retzius cells to 5-HT. In (A), an 800 nA iontophoretic pulse of 5-HT causes a 4 mV hyperpolarization. In (B), the response to the same dose is very much reduced after 6 min in 10-4 morphine, and even after washing for 3 rain in (C), the response to 800 nA 5-HT is still blocked. With further washing (D and E) the 5-HT response returns to its original amplitude. All traces taken from the same cell in the order shown.
Antagonists of the 5-HT response of Hirudo Retzius cells Table 1 lists the compounds examined as potential serotonin antagonists on the Retzius cells of both Hirudo and Haemopis. It is convenient to classify the compounds tested into four groups; compounds active on mammalian 5-HT D or M receptors (Gaddum & Picarelli, 1957); compounds active on other invertebrate 5-HT receptors (Gerschenfeld, 1973; Gerschenfeld & Paupardin, 1973) and other possible antagonists. (a) Control experiment. To ensure that any blockade of 5-HT responses observed was not due to desensitization to repeated doses of agonist, re-
peated iontophoretic doses of 5-HT were administered to the Retzius cells of two preparations. No attenuation of the 5-HT response was observed when the 3 rain drug cycle, as used for the antagonist investigations, was employed. (b) M-receptor antagonists. Morphine antagonized the response of Hirudo Retzius cells to 5-HT but was rather slow acting. Antagonism was observed in only three out of seven preparations incubated for 3 min in the presence of 10-4 M morphine. However, in preparations incubated for 6 min, a reversible blockade of the 5-HT response was observed in two out of three cases. A typical record is shown in Fig. 2. Atropine reduced or
198
P.A. SMITHAND R. J. WALKER Table 1. The effect of 5-HT antagonists on the response of Retzius cells to 5-HT M Receptor Antagonists
No. of obser~a-
Antagonisna of 5 - H T
tio:~ s
Direct Depolarisation effect
Max. dose administered
Morphine
7
++
10"4M/6min
Atropine
9
++
10-4lvI/6min
018
4
] O" 4M/12min
D Receptor Antagonists Dibenamine
6
+++
5 -methoxygramine
5
+
Gramine
5
Methylsergide
8
2 - B r o m o LSD (BOL)
4
++
Sandoz MY25
3
+
Cyproheptadine
3
++÷
10-6M/6min
BW 545 C64
3
if+
10" 6 M / 6 r a i n
10"6Mf5rnin 2 x 10-5M/6rnin 4 x 10-5M/6mln I. 72 x 1 0 - 4 M / 1 2 m i n 10"6M/6min 6 x 10-SM/bmin
A n t a g o n i s t s in I n v e r t e b r a t e s Tryptamine 7
- M e t h y l t r yptarnine
3
10-4M/6min
3
Tubocurarine
2
Bufotenlne*
3
Neo s t i g m i n e *
4
Iproniazid*
3
1 0 - 4 M I 6rain
+
1 O" 4M/6rc~in 2 x 10"4M/1Zmin 5 x 1O'4M/IZmin
+
4 x 10-454/15rain
Other potential antagonists 10-3M/6min
Clonidine
3
Phenyldlguanide
4
+h
10-3M/6min
Mianserin*
4
+
10- 4 M / 6 r a i n
3 ( 3 - A r n l n o p r o p y l ) indoleV
3
2 x 10"4M/12min
a = s u b s t a n c e s t e s t e d oft H a e r n o p i s (all o t h e r s u b s t a n c e s t e s t e d on H i r u d o )
blocked the response to 5-HT in six out of nine cells tested after 3 rain incubation with a 10-4 M solution. Atropine was difficult to wash off and blocking of the response could only be reversed in two preparations. Prolonged incubation with atropine caused the cells to hyperpolarize and, in three out of nine preparations, it increased the duration of the 5-HT response. Offermeier & Ariens (1966) synthesised a compound known as "O18" which was a highly specific M receptor antagonist. This compound failed to antagonize the response to 5-HT in four preparations incubated for up to 12 min in a 10-4 M solution. (c) D-receptor antagonists. Dibenamine blocked the response to 5-HT in four out of six ceils after their incubation, for 3 rain, with low doses (10 -710-6 M). The blockade was usually irreversible. Prolonged incubation in dibenamine caused permanent depolarization of preparations. Thus, although dibenamine was a potent antagonist of 5-HT, its direct action on Retzius cells prevents its use in any further investigations. This direct depolarizing effect was also exhibited by many of the other D-receptor antagonists tested on the Retzius cell, i.e. gramine, 5-methoxygramine,
2-bromolysergic acid diethylamide, Sandoz MY25, cyproheptadine and BW545C64. These compounds failed to antagonize the 5-HT response, but the concentrations at which they could be tested were often limited by their tendency to depolarize the neurones. Table 1 shows the number of experiments performed with each compound and indicates the depolarizing potency of each of these D-receptor antagonists. Methylsergide was tested on eight ceils at concentrations from 4.3 × 10-5 M to 1-72x 10-4 M. This compound showed no tendency to excite cells and did exhibit some anti-5-HT activity. A reversible partial block was observed in three preparations. (d) Antagonists active on invertebrate 5-HT receptors. Tryptamine, 7-methyltryptamine and tubocurarine failed to antagonize the response of Retzius cells to 5-HT even after 6 min incubation with a 10-4M solution (Table 1). Tryptamine hyperpolarized Retzius ceils as would be expected from previous structure activity studies (Smith & Walker, 1974). (e) Other potential antagonists. Clonidine and phenyldiguanide failed to antagonize the response of
Studies on leech 5-HT receptors
2
R.I~
mV
Current
nA
3
~
4
6
7
8
9
199
due to its direct effect on cells, cannot be used for further study. The M receptor antagonists, morphine and atropine, exhibit antagonistic activity to a greater extent that does methylsergide. D-receptor antagonists tend to depolarize the cells.
10
\Control
Antagonists of the 5-HTresponse ofHaemopis Retzius cells
ii'
Fig. 3. The current voltage relationship for a typical leech Retzius cell. Abscissa: current in nA injected into soma. Ordinate: resting potential. Two graphs are shown, one control (V) and one in the presence of 10-I M 5-HT (~7). The calculated value of - 6 9 mV for the 5-HT equilibrium potential (Walker & Smith, 1973) (Es-aT) can be seen to lie within the range where the control and 5-HT V/I curves are superimposed. Retzius cells to 5-HT in concentrations of 10-3 and 10-5 M respectively ( 6 m i n incubations). Phenyldiguanide depolarized Retzius cells in high doses (Table 1). None of the compounds tested act as potent antagonists of the 5-HT response of Hirudo Retzius cells. Dibenamine would be a potent antagonist but,
Morphine, bufotenine, neostigmine, iproniazid, mianserin and 3(3-amino propyl) indole were tested as potential antagonists of the 5-HT response of Haemopis Retzius cells. Morphine was a less effective antagonist on Haemopis than Hirudo. No blockade of the 5-HT response was observed in two out of three cells incubated with 2 x 10-4 M morphine for 12 min. In the third cell potentiation of the 5-HT response occurred. In one cell incubated with 10-3 M morphine for 12 rain, blockade of the 5-HT response occurred. Bufotenine, neostigmine and 3(3-aminopropyl) indole failed to antagonize the response of Retzius cells to 5-HT (Table 1). Iproniazid and mianserin tended to depolarize the Retzius cells but had no effect on their response to 5-HT (Table 1).
Current-voltage relationships The current-voltage relationship for a typical leech Retzius cell is shown in Fig. 3. The neurones exhibit anomalous rectification since the gradient of the curve is recorded with greater hyperpolarization (Kandel & Tauc, 1966). Assuming the Retzius cell to be a sphere of 30 p. radius, the specific membrane resistance for three cells calculated from the initial gradient of their V/I curves is0.725 x 103 f~cm~.
A
B After 3min. in 10-5M Metl~ysergide.
I
W
/
,,4tl
ffJJ C After 3rain. in 10-4M
I, i
te ,
I
--__Jyj
340 nmol 5MeODMT Methysergide.
,,,, I
,0 mV[___.~
I
W D 6rain. later
/f((f( 340 nmol 5MeODMT
Fig. 4. The effect of methylsergide on the response of Retzius cells to 5-methoxy, N,N-dimethyltryptamine (5MeODMT). In (A), 340 nmol 5MeODMT produces a biphasic response. In (B), this response persists after 3 min incubation with 10-s M methylsergide, but after 3 rain in 10-4 M methyl° sergide, a permanent blockade of the response is obtained as shown in (C) and (D) after 6 rain washing. All traces taken from the same cell in the order shown.
200
P.A. SM]TtaAND R. J. WALKER H - Bonding
A
B
matic Ring
Terminal N Binding Site
Fig. 5. (A) A hypothetical scheme for the nature of the 5-HT receptor of the leech Retzius cell. (B) Orientation of 5-HT at the hypothetical receptor site.
studies falls within the range where the 5-HT and control V/I curves are superimposed. The magnitude of the 5-HT response is predicted by the difference between the two curves at constant current (Ginsborg, 1967) and thus the irreversibility of the 5-HT response is to be expected from the superimposed V/I curves.
Further investigations on the depolarizhlg effects of 5-HT agonists and antagonists Previous studies have shown that 5-methoxylated and N-methylated tryptamine derivatives tend to depolarize Retzius cells. (Smith & Walker 1974). Since D-receptor antagonists also depolarize Retzius cells, the possibility of an interaction between the two depolarization responses was investigated. The addition of large doses of 5-methoxy NN-dimethyltryptamine (5MeODMT) to Retzius cell preparations resulted in a depolarization followed by a hyperpolarization. This biphasic response is shown in Fig. 4. Incubation of preparations from Hirudo for 3 m i n with 10 -4 methylsergide irreversibly blocked both phases of the response in two instances. A typical record is shown in Fig. 4. In two other preparations methylsergide either blocked the hyperpolarizing phase or the depolarizing phase of the biphasic response. This indicates that the depolarization of Retzius cells by D-receptor antagonists and N-methylated or 5-methoxylated tryptamine may involve some similar mechanism. DISCUSSION
Fig. 6. Possible orientation of 7-HT within the hypothetical 5-HT receptor.
However, due to the invaginations of the cell membrane, the surface area of the neurone and hence the specific membrane resistance may be as much as fifteen times greater than that of a 30/z radius sphere (Smith, 1975). In the presence of 10 -4 M 5-HT, the specific membrane resistance was reduced to 0.542 × 103 ~-2cm 2. As the voltage on the ordinate of the V/I curves is plotted as an absolute value as in Fig. 3, the equilibrium potential of the 5-HT response is the resting potential at the intersection of the control V/I curve and that in the presence of 5-HT (Ginsborg, 1967). However, in Fig. 3 when the V/I curves assume a shallow gradient, linear relationship, the 5-HT V/I curve and the control V/I curve become parallel and are almost superimposed between - 6 3 and - 7 3 mV and thus no clear indication of ES.aT can be obtained. However, the value of - 69 mV obtained from previous
From the present results, as well as those obtained in a previous study (Smith & Walker, 1974), it is possible to make certain conjectures about the nature of the 5-HT receptor site of Hirudo Retzius cells. Although the model proposed for the receptor in Fig. 5 is extremely hypothetical it serves as a useful framework around which the observed potencies of analogues may be discussed. Since the e.p.m.r, of the analogues tested does not take into account their efficiency (Stephenson, 1956), tryptamine congeners are classified qualitatively and discussed as potent or weak agonists or inactive compounds. The essential features of the hypothetical receptor site are as follows: (I) There exists a large, positively charged or hydrophobic area in the same plane as the indole ring of the agonist specific for aromatic, probably bicyclic, rings with their delocalized electron clouds. Previous studies (Smith & Walker, 1974) have shown that there is no specific binding site for the Jndolic nitrogen. The evidence for this is (a) only compounds with a bicyclic aromatic ring are active, e.g. 5-HT, tryptamine, 3-(2-aminoethyl) 5-hydroxyindene (Smith & Walker, 1974). 3-Phenyl-l-propylamine with its single aromatic ring does not display 5-HT like activity, (b) 6-aminohexanol which
Studies on leech 5-HT receptors has no aromaticity at all is inactive. The model is supported by the e.p.m.r.'s of 5,6 and 5,7 dihydroxytryptamine on Haemopis Retzius cells. These substances are quite active agonists and there is therefore no steric hinderance around the sides of the receptor site and therefore the aromatic binding site must lie above or below the aromatic ring. Also 7-HT is a potent agonist. If molecular models of this compound and 5-HT are compared, the indolic hydroxy groups, terminal nitrogens and six-membered ring parts of the indole ring may be aligned but then the five-membered, nitrogen containing ring occupies a very different position although it can still fit the receptor site as illustrated in Fig. 6. (2) There is a hydrogen binding site at a specific point on the large aromatic binding site. This site is specific for an aromatic hydroxyl group and the electron deficient part of the hydrogen bond is presumably on an amino acid residue of the receptor site. The evidence for this: (a) 5-Fluorotryptamine and 5-chlorotryptamine have fairly potent 5-HT like activity (Smith & Walker, 1974), the halogen moieties of these substances are electron rich and can participate in hydrogen bonding. (b) 6-Aminohexanol is inactive, indicating either the necessity for an aromatic hydroxyl group or that the hydrogen bond alone cannot secure the molecule on the receptor site. (c) Tryptamine, which has no hydrogen bond forming group is much less potent than 5-HT (Smith & Walker, 1974) and 3-phenyl-1-propylamine exhibits no 5-HT like activity.
201
(3) There is a site specific for the terminal nitrogen. This site is approximately 3"5 A. from the large aromatic site and in a precise location compared with the hydrogen bonding site. The evidence for this is: (a) 4-HT and 6-HT are poor agonists (Smith & Walker, 1974) and therefore the position of the H bonding site relative to the side chain must be specially specific for 5-HT. (b) Gramine and 3(3aminopropyl) indole which were tested as potential antagonists (Table 1) did not exhibit 5-HT like activity. These substances have one and three carbons in their side chains respectively (Fig. 1) suggesting that the terminal nitrogen binding site must be the length of two carbon-carbon bonds plus one carbon-nitrogen bond from the aromatic site (i.e. approximately 3.5/~). (c) 5-Hydroxytryptophol is inactive (Smith & Walker, 1974) indicating the importance of the terminal nitrogen. (d) According to experiments on Haemopis, 7-HT can also fit into this suggested conformation of active sites (Fig. 6). Other workers have attempted to formulate models of the 5-HT receptor from structure-activity studies, That proposed in Calliphora by Berridge (1972) closely resembles the present model. Woodruff (1971) has suggested the existence of receptors which may have three hydroxyl binding sites in Helix aspersa. Such receptors are thought to be able to accommodate dopamine, 6-HT and 5-HT. The receptors of the Retzius cells of Hirudo do not correspond with this model as 6-HT (Smith & Walker, 1974) and dopamine (Kerkut & Walker,
Table 2. A comparison of molluscan 5-HT receptors with the annelid H receptors in Hirudo
~ o
Receptor
Response
Ionic P e r m e a b i l i t y change
~ ~
~ ~
~
Molluscan A receptor
Depolarising
Na+( C a + + K + ?)
B
B
B
B
B
B
B
O
B
]3
B
B
O
O
B
O
O
B
B
B
O
O
O
13
]3
B O
-
O
B
O
O
~[olluscan B r~,ceptor
Hyper polarlsing son~etlmes irreversible
~folluscan C receptor
Hyperpolarislng never irreversible
Annelid H receptor
Hyperpolarising irreversible
B
1.
= Brock
K+
CI" Ci"
0
= No effect
F r o m the r e s u l t s with o t h e r e r g o t d e r i v a t i v e s ,
O 10
02
- = not tested
L S D would not be e x p e c t e d to
b l o c k the 5 - H T r e c e p t o r s of H i r u d o . 2.
A n t a g o n i s t t e s t e d on H a e m o p i s .
3.
R e s u l t s of G e r s c h e n f e l d (1973) and G e r s c h e n f e l d and P a u p a r d i n (1973).
4.
R e t z l u s c e l l s did not d e s e n s i t i s e to 5 - H T .
5.
R e s u l t s of C o t t r e l l and M a c o n (1974).
F~5
-
O4 B
202
P.A. SMITHAND R. J. WALKER
1967) are only weak agonists. However, the receptor site in Haemopis, where 6-HT exhibits a greater e.p.m.r, than in Hirudo, may be more similar to that proposed in H. aspersa by Woodruff (1971), though even in this case the potency of 6-HT compared to 7-HT is more than ten times less. In the snail, H. aspersa, Gerschenfeld (1973) and Gerschenfeld & Paupardin (1973) have characterized three 5-HT receptors in terms of their blockade by various antagonists and their ionic mechanisms. The molluscan A, B and C receptors are compared with the present annelid H receptor in Table 2. There is no apparent similarity between any of the molluscan receptors and the annelid receptor. The molluscan C receptor which involves the same ionic mechanism as the annelid receptor is blocked by LSD, tryptamine, atropine, tubocurarine and neostigmine. The only antagonist in this series of components active on the leech 5-HT receptor is atropine, but the compound seems to be fairly non-specific since it also blocks molluscan A and B receptors (Gerschenfeld & Paupardin, 1973). Only molluscan B receptors sometimes fail to exhibit reversibility on hyperpolarization. Gerschenfeld & Paupardin (1973) have suggested that this lack of reversibility is associated with the anomalous rectification properties of the neurones and the probable axonal location of 5-HT receptors. Thus, when the soma is hyperpolarized, its membrane resistance decreases and current leaks out through the somatic membrane without influencing the resting potential in the axon where the 5-HT response occurs. A similar mechanism may explain the lack of reversibility of the Retzius cell 5-HT response (Walker & Smith, 1973). The present study has demonstrated the anomalous rectification properties of these neurones and it is probably that the 5-HT receptors lie on the axon (Smith, 1975). It is possible that the response of Retzius cells to N-methylated and 5-methoxylated tryptamines may not be mediated by the receptor depicted in Fig. 5 (Smith & Walker, 1974), although these compounds act by a similar mechanism to D-receptor antagonists. Three theories may be advanced to account for the effects of N-methylated and 5-methoxylated tryptamines on Retzius cells. (1) It is possible that these compounds combine with 5-HT receptor site in some different combination from 5-hydroxylated analogues. This may promote a depolarization rather than a hyperpolarization. Crystallographic studies on lysozyme (Beddell et al., 1970) have shown that small changes in the substrate molecule can very much alter its mode of combination at the active site. (2) There may exist two tryptamine receptors on the leech Retzius cell. One receptor would be that shown in detail in Fig. 5 which may resemble the mammalian M-receptor since it is blocked by atropine and morphine. 5-HT would be the best agonist and would produce pure byperpolarizations at this
receptor. The other receptor may resemble the mammalian D-receptor; its stimulation, which is best brought about by N-methylated and 5-methoxylated tryptamine, may result in biphasic responses or depolarizations. D-receptor antagonists may act as partial agonists on this receptor and 5-HT may be an antagonist since preparations often do not respond to 5-methyoxylated or N-methylated compounds after application of this substance (Smith & Walker, 1974). (3) As production of excitation or a biphasic response requires high doses of agonist (Smith, 1975) the most likely explanation for the action of Dreceptor antagonists and 5-methoxylated and Nmethylated tryptamines is that they may have some non-specific interaction with the cell membrane. This may be associated with the exceptional charge transfer ability of these compounds (Snyder & Merril, 1965). Acknowledgements--We are grateful to the following for gifts of compounds: Professor E. J. Ariens, Dr. M. J. Berridge, Messrs. Boehringer Ltd., Messrs. Organon Ltd.; to S.R.C. for a training grant to P. A. S.; and to Mr. M. D. King for useful discussions. REFERENCES AMIN A. H., CRAWFORD T. W. • GADDUM J. H. (1954)
The distribution of substance P and 5-hydroxytryptamine in the central nervous system of the dog. J. PhysioL, Lond. 126, 596-618. BEDDELL C. g., MOULT J. & PHILLIPS D. C. (1970) Crystallographic studies on the active site of lysosyme. In Molecular Properties of Drug Receptors (Ciba Foundation). (Edited by PORTERR. & O'CONNORM.), pp. 85-112. Churchill, London. BERRIDGEM. J. (1972) The mode of action of 5-hydroxytryptamine. J. exp. Biol. 56, 311-321. COTTRELL G. A. & MACON J. B. (1974) Synaptic connexions of two symmetrically placed giant serotonin containing neurones. J. Physiol., Lond. 236, 435-464. GADDUMJ. H. & PICARELLIZ. P. (1957) Two kinds of tryptamine receptors. Br. J. Pharmac. Chemother. 12, 323-328. GERSCHENFELO H. M. (1973) Chemical transmission in the invertebrate central nervous systems and neuromuscular junctions. Physiol. Rev. 53, 1-119. GERSCHENFELDH. M. & PAUPARDIND. (1973) Actions of 5-hydroxytryptamine on molluscan neuronal membranes. In Drug Receptors--A Biological Council Symposium (Edited by RANG H. P.), Macmillan, London. GERSCHENFELDH. M. & PAUPARDIN-TRITSCHD. (1973) Excitatory and inhibitory monosynaptic actions mediated by a serotonin containing neurone in Aplysia californica. J. Physiol., Lond. 234, 28P. GERSCHENFELD H. M. t~ STEFANI E. (1968) Evidence for an excitatory transmitter role of serotonin in molluscan central synapses. Adv. Pharmac. 6A, 369-392. GINSBORGB. L. (I 967) Ion movements in junctional transmission. Pharmac. Rev. 19, 289-316. KANDEL E. R. & TAUC L. (1966) Anomalous rectification in the metacerebral giant cells and its consequence for synaptic transmission. J. Physiol., Lond. 183, 28%304.
Studies on leech 5-HT receptors K~RKtrr G. A. & WALKER R. J. (1967) The action of acetylcholine, dopamine, 5-hydroxytryptamine on the spontaneous activity of the cells of Retzius of the leech, Hirudo medicinalis. Br. J. Pharmac. Chemother. 30, 644-654. OFFERMEIER J. • ARIENS E. J. (1966) Serotonin I - Receptors involved in its action. Archs int. Pharmacodyn. 164, 192-215. RETZIUS G. M. (1891) Zur Kenntnis des centralen Nervensystems der Wiirmer. Biol. Unters (NF) 2, 1-28. SMrra P. A. (1975) Neurophysiological and neuropharmacological studies on the Retzius cells of the leeches Hirudo medicinalis and Haemopis sanguisuga. Ph.D. thesis, University of Southampton. SMITH P. A. • WALKER R. J. (1974) The action of 5hydroxytryptamine and related compounds on the activity of the Retzius cells of the leech, Hirudo medicinalis. BE. J. Pharmac. 51, 21-27. SNYDER S. H. & MERRIL C. R. (1965) A relationship between hallucinogenic activity of drugs and their electronic configuration. Proc. natn. Acad. Sci. U.S.A. 54, 258-265. STEPHENSON R. P. (1956) A modification of receptor theory. Br. J. Pharmac. Chemother., 11, 379-393. TWAROG B. M. & PAGE I. H. (1953) Serotonin content of some mammalian tissues and urine and a method for its determination. Am. J. Physiol. 175, 157-161.
203
WALKERR. J., RALPH K. L., WOODRUFFG. N. & KERKUT G. A. (1972) Evidence for acetylcholine and 5-hydroxytryptamine as excitatory transmitters onto a single neurone in the brain of the snail, Helix aspersa. Comp. gen. Pharmac, 3, 52-60. WALKERR. J. & SMITH P. A. (1973) The ionic mechanism for 5-hydroxytryptamine inhibition on Retzius cells of the leech, Hirudo medicinalis. Comp. Biochem. Physiol. 45A, 979-993. WALKER R. J. & WOODRUFF G. N. (1972) The effect of bufotenine, melatonin, psilocybin and related compounds on the 5-hydroxytryptamine excitatory receptors of Helix aspersa neurones. Comp. gen. Pharmac. 3, 27-40. WELSH J. H. (1968) The distribution of serotonin in the nervous systems of various animal species. Adv. Pharmac. 6A, 171-188. WELSH J. H. & MOORHEAD M. (1960) The quantitative distribution of 5-hydroxytryptamine in the invertebrates, especially in their nervous systems. J. Neurochem. 6, 146--169. WOODRUFF G. N. (1971) Dopamine receptors: a review. Comp. gen. Pharmac. 2, 439-455. Key Word Index--5-HT; receptors; leech; Hirudo; Haemopis; morphine; atropine; antagonist; agonist.