Evidence for an amine receptor on the Retzius cells of the leeches Hirudo Medicinalis and Haemopis Sanguisuga

Comp. Biochem. PhysioL Vol. 67C, pp. 159 to 166

0306-4492/80/110[-0159502.(~)/0

© Pergamon Press Lid 1980. Printed in Great Britain

E V I D E N C E F O R A N A M I N E R E C E P T O R O N THE R E T Z I U S CELLS O F THE LEECHES H I R U D O MEDICINALIS AND HAEMOPIS SANGUISUGA A. J. SUNDERLAND,*L. D. LEAKE Department of Biological Sciences, Portsmouth Polytechnic, Portsmouth PO1 2DY, England and R. J. WALKER Department of Neurophysiology, School of Biochemical and Physiological Sciences, University of Southampton, Southampton, SO9 3TU, England (Received 25 March 1980) Abstract--1. The hyperpolarizing responses of Retzius cells from Hirudo medicinalis and Haemopis

sanguisuga to 5-hydroxytryptamine (5-HT) and dopamine (DA) were examined and the effectiveness of 9 putative antagonists determined. 2. DA is less potent than 5-HT (4.1 x in Haemopis; 20 x in Hirudo). Dose/response curves and time-to-peak data were compared. 3. Cross-densensitization occurs in both species. 4. Neither the DA nor the 5-HT responses could be selectively blocked in either species. Atropine and morphine are the most potent antagonists. 5. It is concluded that DA and 5-HT act at the same receptor on the membrane of leech Retzius cells.

INTRODUCTION

lion but have been unable to detect any other catecholamines. Possible metabolic pathways for DA are There is evidence for the presence of the naturally indicated by the experiments of Stuart et al. (1974) occurring biogenic amines 5-hydroxytryptamine who found that quantities of tyramine, DA and (5-HT) and dopamine (DA) in the leech central neroctopamine were synthesized by segmental ganglia vous system. In each segmental ganglion there are incubated in tyrosine. Of these amines only DA fluorseven cells which show the yellow fluorescence esces when subjected to the Falck-Hillarp technique. characteristics of 5-HT when examined using the DA is not found ~n the Retzius cells (Osborne et al. Falck-Hillarp technique (Kerkut et al., 1967; Ehinger 1972). et al., 1968; Marsden & Kerkut, 1969; Rude, 1969). Both dopamine and 5-HT hyperpolarize the memMicrochemical analysis of the two largest of these brane potential of the Retzius cells (Kerkut & Walker, neurons, the Retzius cells, shows that they contain 1967). The hyperpolarization is, in both cases, the 1-4 pmol of 5-HT per cell and between them account result of an increase in the permeability of the memfor 40% of the 5-HT to be found in the ganglion brane to chloride ions (Walker & Smith, 1973; Sun(Rude et al., 1969; McCaman et al., 1973; McAdoo derland et al., 1979). & Coggeshall, 1976). It has been demonstrated that This paper investigates the characteristics of the the segmental ganglion can synthesise 5-HT from dopamine and 5-HT responses of the Retzius cells tryptophan (Hildebrand et al., 1971; Sargent, 1977; , and the effectiveness of a range of compounds as McAdoo, 1978) and that aromatic acid decarboxylase, specific antagonists of these responses. The impli5-hydroxytryptophan and tryptophan are present in cations for the form of the 5-HT and DA receptors the Retzius cells (Coggeshall et al., 1972; Osborne et are discussed. al., 1972). The evidence for DA is less substantial. Two cells in MATERIALS AND METHODS the anterior lateral roots of the ganglion show the green fluorescence characteristic of the catecholThe two species of leech (Hirudo medicinalis and Haeamines when stained using the Falck-Hillarp tech- mopis sanguisuga) were kept in 4% leech Ringer in distilled nique (Ehinger et al., 1968; Rude, 1969). The axons of water at 12°C. Intracellular recordings were made from the these monopolar neurons run into the ganglion and Retzius cells of isolated ganglia as described previously divide sending one branch in an anterior direction (Sunderland et al., 1979). The normal leech Ringer used and the other posteriorly (Rude, 1969). McCaman et contained; NaC1 115 mM, KCI 4 mM, CaCI2 2 mM, Glual. (1973) and McAdoo & Coggeshali (1976) have cose 10 mM, Tris-chloride buffer l0 mM, pH 7.4 (Kuffler detected small amounts of DA in the segmental gang- & Potter, 1964). Magnesium (20 mM) Ringer was made by reducing the NaCI concentration to 95 mM and including both MgCI2 (10mM)and MgSO,, (10mM). All the drugs were made up in and administered in the same Ringer as * A.J.S. is an SRC Research Fellow. that bathing the preparation at the time of application. 159

A.J. SUNDERLANDet al.

160

Agonists were applied in 1 ml aliquots to the preparation contained in a 10 ml bath. The dose-response curves for dopamine and 5-HT were plotted from the averaged data from 10 experiments (Haemopis) and 18 experiments (Hirudo). Increasing doses of agonist were applied at 5 min intervals and the preparation was washed copiously between applications. Both DA and 5-HT were applied to the same preparations. A series of experiments was performed to determine the relative effects of densensitization of the preparation to either DA or 5-HT on the responses produced by the two agonists. Approximately equipotent doses of DA and 5-HT were determined for a preparation, which was then subjected to repeated applications (ca. 20) of high concentrations (2.10-4-5.10 -3 M) of either 5-HT or DA. Subsequently the responses to the "equipotent'" doses of DA and 5-HT were redetermined. The time courses of the responses to DA and 5-HT were determined from the permanent records made during the above experiments. Time to peak of the response and time to half peak were measured from the onset of hyperpolarization. The effectiveness of potential antagonists was examined on preparations which gave a clear response to a known dose of agonist. The normal Ringer bathing the preparation was then exchanged for Ringer containing the antagonist and the preparation incubated for 10 rain before the same dose of agonist was reapplied. The reversibility of the effective antagonists was assessed by the application of a third dose of agonist applied after the antagonist had been removed from the preparation by prolonged washing with normal Ringer. If the preparation showed less than a full recovery the washing was repeated and the agonist applied again. This was repeated until the response had either fully recovered or remained constant. The drugs used were:- 5-hydroxytryptamine creatinine sulphate (Sigma), Dopamine hydrochloride (Sigma), Atropine sulphate (BDH), E~gometrine maleate (Sandoz, gift), Fluphenazine hydrochloride (Squibb), Methysergide bimaleate (Sandoz), Metaclopromide monohydrochloride (Merk, Sharp & Dohme), Morphine hydrochloride (BDH),

Aa

I!'

Phentolamine mesylate (CIBA), Propanolol hydrochloride (ICI, gift) and Strychnine hydrochloride (BDH). RESULTS Dopamine and 5-HT both hyperpolarize the Retzius cells and this response persists in 20 mM magnesium Ringer (Fig. 1). The maximum responses of the Retzius cells of Haemopis to both dopamine and 5-HT are the same and the two dose-response curves are similar (Fig. 2). The displacement of the dopamine curve to the right indicates that it is less potent than 5-HT. A comparison of the doses required to produce a half maximal response (1.3 × 1 0 - S M 5-HT: 5.3 x 10-SM DA) shows that dopamine is 4.1 times less potent than 5-HT. The Retzius cells of Hirudo medicinalis have different maximal responses to dopamine and 5-HT, that to dopamine being the smaller by 2 mV (Fig. 2). The two dose-response curves are approximately parallel for most of their length differing only at the higher concentrations where they approach different maxima. The difference in potencies is greater than in Haemopis. The ratio of the doses required to produce a half maximal response (1.3 × 10-SM 5-HT: 2.6 x 1 0 - a M DA) shows that dopamine is 20 times less potent than 5-HT. Haemopis Retzius cells showed equal desensitization of the responses to equipotent doses of 5-HT and DA after repeated doses of a high concentration of either agonist (5-HT in 3 preparations, DA in 3 preparations). Similarly, in Hirudo repeated doses of a high concentration of dopamine attenuated the responses to equipotent doses of both agonists (3 preparations). When the concentrations of the two agonists required to give equal responses in the Hirudo Retzius

IL I 52 pM 5-HT

I

I

~ II

4b wash

4b

52 ~JM 5-HT

wash

J

2OmV

20s

Bs

~ A a A 4 4 . . L ~

..a.~.~~~'

110 IJM DA

w S sh

110 ~M DA

4b wash

,

I!

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Fig. 1. Traces from an Hirudo Retzius cell showing that the hyperpolarizing responses to 5-HT (Aa) and Da (Ba) remain when the preparation is bathed in 20 mM magnesium Ringer (Ab, Bb).

161

Amine receptor in Retzius cells

Haemop_is 10-

8E 6-

~:4-

2-

IP" IB'=~~ O

•

10-8

10-6

i

10-4

10-2

Dose (M)

Hirudo lO-

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6 .

rr4-

2-

•

L

o

10-8

~i-

i

i

r

10-6

1'0-4

I

I

10-2

Dose (M)

Fig. 2. Plots of the dose/response curves for 5-HT (O) and DA (11) on the Retzius cells of the two leech species. The plots represent the average of 10 experiments on Haemopis and 18 on Hirudo.

cells were within a factor of ten, repeated applications of a high concentration of 5-HT densensitized both responses (3 preparations). However, when the difference in concentration was greater than ten, repeated app!ications of 5-HT densensitized the test 5-HT response but not that to DA (3 preparations). A comparison was made of the time-courses of the response to those concentrations of the two agonists

required to give half maximal responses in the Retzius cells of the two species (Table 1). The time-course of the response to dopamine and 5-HT in Haemopis and to 5-HT in Hirudo were similar, the time to half maximum being 3-4 sec and that to maximum being 1(~15 sec. In Hirudo the DA response was much slower, hyperpolarizing to half maximum in 10 sec and to maximum in 36.5 sec.

Table 1. The average times to half peak and to peak of the responses of both Hirudo and Haemopis Retzius cells to half maximum doses of 5-HT and DA Dose required to give a half maximal response to the agonist (M) Time to half peak of the response(s) Time to the peak of the response(s) Number of observations

Hirudo

Haemopis

5-HT 1.3 10 -s

Dopamine 2.6 10 -4

5-HT 1.3 10 -5

Dopamine 5.3 I0 -5

4 ___0.6

10 __+1.7

3.3 + 0.7

3.2 + 0.3

14 4- 1.9

36.5 ___5.3

10.3 + 1.3

14.6 ___ 1.5

6

12

7

25

A.J. SUNDERLANDet al.

162

Table 2. The effect of 9 putative antagonists on the responses of the Retzius cell to 5-HT and DA. The effectiveness of the antagonists is graded: + + + - effective in >90~o of the preparations examined; + + = effective in > 50"~,, <90"~, of preparations examined; + = effective in <50% of preparations examined, and - = no antagonism in any preparations examined. Direct effect

Antagonist 5-HT antagonists Atropine Morphine Methysergide DA-antagonists Fluphanazine Metaclopramide Ergometrine Adrenergic antagonists Phentolamine Propranolol Strychnine

Antagonism of 5-HT No. expt. effectiveness

Antagonism of DA No. expt. effectiveness

Max. dose administered

H H --

15 5 3

+ + + + + --

17 7 4

+ + + + + + --

1.4 10 -4 M 2.3 10 -4 M 1.4 10 -a M

H --

7 7 9

+ + +

22 5 9

+ + +

2.2 10 -4 M 3.0 10 -4 M 2.3 10 -4 M

D -H

3 4 5

--+ +

5 3 5

--+ +

2.7 10- 4 M 1.7 10 -7 M 1.2 10 -4 M

Table 2 shows the 9 c o m p o u n d s examined as potential antagonists of 5-HT and D A on the Retzius cells of b o t h Haemopis and Hirudo. N o species differences were found in the effectiveness of the antagonists. The c o m p o u n d s that showed antagonist activity were tested against b o t h 5-HT a n d DA on the same preparation. A given concentration of a particular antagonist was found to have the same effect on 5-HT and D A responses of the same size. It would antagonize neither response or b o t h responses to the same extent. Consequently, those of the compounds that were potent antagonists of 5-HT were potent antagonists of DA and the inactive c o m p o u n d s were inactive against b o t h agonists. The most potent blockers were the 5-HT antagonists atropine (Fig. 3) and m o r p h i n e (Fig. 4), but they also hyperpolarized the cells, morphine more so than atropine. Methysergide was inactive at the concentrations used. The DA antagonists blocked the responses but were much

weaker than the 5-HT antagonists. Fluphenazine blocked the DA response but not the 5-HT response in one preparation of Haemopis but in subsequent experiments either b o t h or neither response was blocked. With the exception of fluphenazine, which hyperpolarized the cells, the d o p a m i n e antagonists had n o direct action. The specific ~-and fl-adrenergic antagonists were ineffective but phentolamine did depolarize the cell. Strychnine blocked b o t h 5-HT and D A responses. Four antagonists were tested against the direct hyperpolarizing effect of morphine. The response to m o r p h i n e was antagonized by atropine in 6 preparations (Max. dose administered = 1.1 × 1 0 - 4 M ) a n d by fluphenazine in 4 of 6 preparations (Max. dose administered = 1.1 × 10- 4 M). P h e n t o l a m i n e and propanolol were each tested against the m o r p h i n e response in 6 preparations (Max. dose administered = 1.3 x 1 0 - 4 M and 1.7 x 1 0 - 4 M respectively a n d

A

4b wash

160pM DA

B After 10 minutes in 35 J:M Atropine

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C

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wash

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Fig. 3. The effect of atropine on the response of the Hirudo Retzius cell to DA. A, the effect of DA; B, after 10 min in atropine the same dose of DA gives a reduced response. After washing (C), the DA response returned.

Amine receptor in Retzius cells

163

A

130iJM 5-HT

4k

260 ~M OA

wash

wash

B After 10 mblutes in 160 ~M moq)hine " 20s ~20mV

4k 1301~M 5-HT

C

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4b

4k 260 ~M DA

wash

4b wash

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db 130 ~IM 5-HT

wash

260 ~111 DA

wash

Fig. 4. The effect of morphine on the response of the Hirudo Retzius cell to 5-HT and DA. A, 5-HT and DA both caused equal hyperpolarization. B, after 10 min in morphine the cell was repolarized artificially to approximately its original resting potential (this was necessary because morphine is a potent agonist and had hyperpolarized the cell) and the same doses of 5-HT and DA reapplied. The responses were abolished. After washing (C) the normal responses to 5-HT and DA were re-established. were found to be ineffective with the exception of a single preparation from Haemopis in which phentolamine showed slight antagonist activity. DISCUSSION

The persistence of the responses of the Retzius cell to 5-HT and DA in 20 mM magnesium Ringer, which has been shown to block chemical synapses in the leech central nervous system (Stuart, 1970), demonstrates that the agonists are acting directly upon the Retzius cell and not via an interneuron. The question then arises as to whether 5-HT and DA are acting on separate receptors or a single common receptor. In determining this, the use of specific antagonists is of great value. Of the 9 antagonists tested against the 5-HT and DA responses atropine was the most potent. Its antagonism of the serotonin response (Smith & Walker, 1975) was confirmed but, in contrast to its activity in Helix neurons (Walker et al., 1968) it proved to be an equally effective antagonist of the DA response. Morphine, previously shown to be an antagonist of 5-HT both in Helix pomatia (Cottrell et al., 1974) and in the leech Retzius cell (Smith & Walker, 1975) was an equally potent antagonist of both responses. Methysergide, also an antagonist of 5-HT in the leech Retzius cells (Smith & Walker, 1975) and Helix pomatia neurones (Cottrell et al., 1974), antagonizes the DA-induced stimulation of the cockroach salivary glands (Ginsborg et al., 1976) and the DA-induced relaxation of the Mytilus anterior byssus retractor muscle (Twarog et al., 1977). However, at the concentrations used in these experiments methysergide was ineffective on R cells. The putative DA antagonists have been shown to be effective in a number of other preparations. Batta et al. (1977) found that fluphenazine and metaclopromide antagonised the DA-induced hyperpolarizing response in neurons of Helix aspersa. Metaclopramide was also a good antagonist of the dopaminergic inhi-

bition of the heartbeat of Chione stachburyi (Gordon & McGregor, 1976). Ergometrine has been used effectively against the dopaminergic hyperpolarization of many molluscan neurons (Walker et al., 1968; Kerkut et al., 1969; Woodruff et al., 1970; Ascher, 1972; Berry & Cottrell, 1975), and to antagonize DA at the molluscan gill (Malanga, 1975) and insect salivary glands (Ginsborg et al., 1976). Fluphenazine, metaclopramide and ergometrine are weak antagonists of the DA response in the leech Retzius cell and also of the 5-HT response. With the exception of one unrepeatable experiment on Haemopis, in which fluphenazine selectively blocked the DA response, the antagonism that was strong enough to block the response to One of the agonists blocked the response to the other. The ~t-adrenergic antagonist, phentolamine, is effective at a number of dopamine receptors in the invertebrates, e.g. neurons of Helix aspersa (Walker et al., 1968) and Aplysia (Ascher, 1972). The fl-adrenergic antagonist, propranolol, is not as effective as it fails to block the DA-induced hyperpolarization of Helix aspersa neurones (Walker et al., 1968). However, both phentolamine and propranolol have blocked the DAinduced stimulation of the isolated salivary glands of the ixodid ticks (Kaufmann, 1977). Neither antagonist was effective against either DA or 5-HT in this preparation which suggests that the receptors at which the agonists act are not of the classical adrenergic type. Strychnine blocks the depolarizing response to DA in Aplysia neurones (Ascher, 1972). It has a direct effect on leech Retzius cells causing them to hyperpolarize by increasing the conductance of the membrane to chloride ions (Prichard, 1971), and acts at a different receptor to 5-HT on the Retzius cell membrane. Walker & Smith (1978) were able to block the 5-HT response with mianserin but not the strychnine response. In this study strychnine was found to block both the 5-HT and DA responses. As strychnine, like 5-HT and DA, exerts its effect by increasing the permeability of the membrane to chloride ions it is poss-

164

A. J. SUNDERLAND et al.

ible that it blocks the 5-HT and DA responses not by obstructing the receptors but by influencing the chloride ionophores and gradient. If the amines and strychnine operate a common set of chloride ionophores then the application of DA or 5-HT to a preparation which has been incubated in strychnine will produce a much reduced response because the ionophores will already have been opened. Incubation of the preparation in strychnine will in any case result in a chloride flux and a change in the chloride concentration gradient. Subsequent application of either amine will produce a much reduced response. It is possible that morphine, which is a potent agonist, exerts its antagonist activity like strychnine, by influencing the chloride ionophores and the chloride gradient. However, like 5-HT and DA it is antagonized by atropine and fluphenazine. Both atropine and fluphenazine have weak agonist activity and are, therefore, unlikely to be able to induce a chloride flux of sufficient magnitude to prevent a response to subsequent applications of either DA, 5-HT or morphine. Consequently it would appear that atropine and fluphenazine act directly at the DA and 5-HT receptors. As morphine is also antagonized by atropine and fluphenazine it too must be acting at these receptors. Thus it is not possible to separate two receptors using these nine antagonists. Neither is it possible to categorize the receptor as either serotonergic or dopaminergic on the strength of the efficacy of the antagonists. The properties of the responses of the Haemopis Retzius cells to dotSamine and 5-HT are almost identical. The time taken by the cells to reach the peak of their responses to dopamine is only slightly slower than that to reach peak response to 5-HT. The times to half peak are identical. Only a small difference in potency separates the dose-response curves. The fact that the properties of these responses in Haemopis differ so little, coupled with the results of the antagonist experiments, suggests that the two amines are probably acting at the same receptor. Such a conclusion is supported by the desensitization experiments which show that a preparation desensitized to 5-HT is also desensitized to dopamine, and vice-versa. The resting potential returns to the same level between applications of the agonists so the desensitization is unlikely to be the result of a depletion of internal chloride, because the cells respond to changes in the chloride concentration gradient with a shift in resting potential (Leake et al., 1977). The differences in properties of the responses of Hirudo Retzius cells to DA and 5-HT could be taken to indicate that the agonists are acting at different receptors or that DA is a partial agonist at a common receptor. The single receptor theory is supported by the results of the antagonist experiments. The fact that the response to DA can be desensitized by repeated applications of 5-HT (3 out of 6 preparations) and that to 5-HT can be desensitized by repeated applications of DA (all 3 preparations) also favours the argument for the agonists acting at a single receptor. If the agonists are acting at a single receptor the smaller maximum response for dopamine shown in the dose-response curves must mean that it is a partial agonist at that receptor. This conclusion is supported indirectly by the time-to-peak data.

According to the theory of Paton (1961) the stimulus produced by an agonist is determined by its nett rate of association with the receptor. The nett rate of association depends upon the ratio of the rates of association and dissociation. Assuming similar availability at the receptor the rapid response of the Hirudo Retzius cells to 5-HT (14 sec) indicates that drugreceptor complexes were being formed at a much more rapid rate than those formed by DA (response time = 36.5 sec). Paton (1961) suggested that all compounds which act at a particular receptor fall on a continuum from rapidly acting agonists to persistent antagonists, depending upon their rates of association and dissociation. The interpretation of the time-to peak data suggested above would imply that although 5-HT might be at the rapid agonist end of the continuum DA is not. The slower rates suggest a position on the continuum slightly towards the persistent antagonism end; an area in which the slower kinetics of the response are the result of a proportion of the available receptors being associated as drug receptor complexes, and therefore, unavailable. Such an area ol the continuum is occupied by the partial agonists. There are precedents for a joint 5-HT-DA receptor in the invertebrates. Malanga (1975) proposed such a receptor on the frontal cilia of the Mytilus gill and Twarog & Cole (1972) have demonstrated that 5-HT and DA both act at the receptor which mediates relaxation in the anterior byssus retractor muscle of Mytilus. A detailed proposal for a joint 5-HT-DA receptor on some Helix aspersa neurones has been made by Woodruff (1971). IPSP's have been recorded from the Retzius cells of Haemopis (Smith et al., 1975). These IPSP's were abolished (in a single preparation) by 50% chloride Ringer which suggests that they are chloride mediated as are the responses to 5-HT and DA (Walker & Smith, 1978, Sunderland et al., 1979). In other preparations the IPSP's were blocked by morphine, which is a good antagonist of both 5-HT (Smith & Walker, 1975) and DA. The IPSP's therefore, may well be the result of an aminergic synaptic input to the Retzius cells. This input cannot come from any of the 5 remaining serotonergic cells in the same ganglion as Lent & Frazer (1977) have shown that the connections to the Retzius cells from these neurons are electrical and not chemical. There is as yet no evidence for a dopaminergic input but the axons of the monopolar neurons which may well contain dopamine (Rude, 1969) pass through the parts of the neuropile that contain Retzius cell branches. There is no information as yet about any connections between the serotonergic cells of adjacent ganglia except that there is not a connection between the Retzius cells of adjacent ganglia (Gerasimov, 1967). It is concluded that there is considerable evidence that DA and 5-HT act at a single amine receptor on the leech Retzius cells. SUMMARY

The responses of Retzius cells from the leeches Hirudo medicinalis and Haemopis sanguisuga to 5-HT and DA were examined and the effectiveness of 9 putative antagonists determined. In Haemopis DA is 4.1 × less potent than 5-HT. The responses to both agonists

Amine receptor in Retzius cells develop over the same time-course and reach the same maximum value. A preparation desensitized to one agonist is desensitized to the other In Hirudo DA is 20 × less potent than 5-HT. The response to DA is slower to develop and has a smaller maximum value than that to 5-HT. Desensitization of a preparation to DA also desensitizes it to 5-HT. Desensitization to 5-HT is accompanied by desensitization to DA when the concentrations of the test doses of the two agonists are within a factor of ten. Of the 9 putative antagonists tested on the Retzius cells of both leech species, atropine and morphine are the most potent; strychnine is effective; fluphenazine, metaclopramide and ergometrine are very weak; and phentolamine and propranolol are ineffective. N o n e of the antagonists selectively blocked either the DA or the 5-HT responses. It is concluded that DA and 5-HT act at the same receptor on the membrane of leech Retzius cells.

REFERENCES

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