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Pharmacological control of muscular activity in the sea urchin larva. I. Effects of nicotinic and muscarinic agents
Camp Bmchem. Physiol. Vol. 94C, No. 1, pp. l-14, Prmted III Great Bntam
1989
0306-4492/89 53 00 + 0.00 Pergamon Press plc
PHARMACOLOGICAL CONTROL OF MUSCULAR ACTIVITY IN THE SEA URCHIN LARVA. I. EFFECTS NICOTINIC AND MUSCARINIC AGENTS
OF
TRYGGVEGUSTAF.WN The Wenner-Gren
Institute,
Biologihus
F3, University (Received
25 Muy
of Stockholm,
S-106 91 Stockholm,
Sweden
1988)
Abstract-l. The muscular system of the sea urchin pluteus is stimulated by nicotinic agents. Excessive stimulation is followed by paralysis of the most powerful muscular strands. The effects are counteracted by antinicotmic agents. 2. Muscarimc agents keep the activrty low. calm down initially unquiet larvae, and counteract the nicotinic effects. 3. The effects are quickly elicited and in general are also quickly reversible in sea water, whtch indicates that most of the receptors mvolved occur at the larval surface. 4. It is suggested that the nicotinic and muscarinic signals control certain ionic fluxes in opposite directions, and that the stimulatory effect on the most powerful muscular strands is amplified by a monoaminergic system.
INTRODUCTION The muscular system of the mature pluteus has been used as a model that may shed some light on the pharmacological signals which appear to control the morphogenetic movements in the embryo as well as its ciliary activity, cf. Gustafson and Toneby (1971) and Soliman (1983a, b; 1984a, b). An advantage of the system and technique used is that the reactions are quick and distinct, and that the reversibility of the effects of various agents and their interaction can be investigated. In addition, several types of contractile activity can be studied. This system may also elucidate some properties of a primitive pacemaker system. This may be particularly important as the vertebrates and the echinoderms may have a common ancestor. The anatomical organization of the system is described in Fig. 1 which also defines the activities the twitch-like L-movements, concerned, the peristaltic S-movements made up of consecutive Ltwitches, the powerful C-activity and the closely related P-movements. It also shows some changes in the ciliary system at the animal plate or velum; St indicates ciliary paralysis. The different muscular movements can be combined in various ways and appear to be pacemaker-controlled. One may say that the pacemaker-signals may choose from a menu of various activities. Biochemical and histochemical studies show that the sea urchin larva contains cholinesterases, acetylcholine, and various monoamines, cf. Augustinsson and Gustafson (1949), Buznikov et al. (1964, 1968), and Toneby (1977, 1980). The aim of the present program is to further investigate this system, not least the mechanism of action of the cholinergic and monoaminergic agents. Preliminary results on the muscular effects of a number of pharmacological agents have been reported earlier (Gustafson et al.,
1972a, b). A detailed account of the stimulatory and paralytic effects of carbachol and physostigmine has also appeared (cf. Fig. 9 in Gustafson and Treufeldt, 1981 with Fig. 3 in Treufeldt and Gustafson, 1981). In the present paper the effects of a series of cholinergica have been studied in order to elucidate to what extent their different responses are mcotinic or muscarinic or reflect a nicotinic-muscarinic interaction. The actions of catecholamines, serotonin, cyclical nucleotides, and their relation to the flux of Ca*+ and K+ will be discussed in forthcoming reports in this series. MATERIALSAND METHODS The material used is the same as in our earlier investigatrons i.e. 3 day olu plutei of Psammechinus miliaris, of both S- and the Z-form living at different depths and therefore at different salinities, 34-30 respecttvely around 25 promtlle. The technique applied was described in earlier papers (Gustafson et al., 1972a; Gustafson and Treufeldt, 1981). RESULTS The description of the results may appear tedious, in particular as the muscular system is rather intricate, and the terminology may look exotic. However, most of the facts are important for the final interpretation of the muscular control mechanism, cf. the discussion. Diagrams
The muscular effects of the agents concerned are illustrated in diagrams where the frequency of different movements, i.e. the number of movements during each of the 15 min long periods of recording (Y-axis), is plotted against time in hr after the control period (X-axis) except in Fig. 12d. Each diagram refers to a single experiment. The application of the
TRYGGVE GUSTAFS~N
Fig. 1.Anatomy of the contracttle system of a 3-day old plutcus of Psa~n~ze~~zzn~.~ mrbarzs and the variation m the state of the ctlia at the antmal plate from normal to paralyws: the L-, S-, C-, and P-nlovements are defined by arrows; upon strong stimulatton the C-movements may be tetanic (see the symbol T in the dragrams) and strong tetanic activity IS followed by paralysis of the C-movements; the N-movements first appear in 4-day old larvae; St mdlcates cihary paralysis Modtfied after Gustafson and Treufeldt (1981)
agents concerned is indicated by arrows. The C-activity is marked by round dots (Fig. 12d being an exception), the S-activity by + signs, the L-activity by rhombic dots. and the P-activity by triangles. Intense tetanic C-activity is marked by a large T; the tetanic intensity is often so strong that individual C-movements cannot be recorded. A somewhat weaker tetanic C-activity is indicated. Agents with pure nicotinic uction The effects of these agents are summarized m Table IA. a. At high concentrations of nicotine, 10 -“-IO-” M, occasionally also at 5 x IO-‘M, there is an immediate burst of strong, tetamc C-activity, instantly followed by permanent C-paralysis, There is also a strong activation of the S- and L-movements which, at the highest concentration, is followed by low activity or even by paralysis (Fig. 2a). At 2 x IO-’ M the initial C-activity is weak but followed by a strong, progressive C-activation (Fig. 2b). At lo-‘M, only a moderate stimulation may be seen The C-paralysis is accompanied by an instant change in the state of the ciha, in particular at the animal plate. The cilia thus become stiff and immobilized. a change that may be denoted “ciliary paralysis”, in earlier reports called stiffening, abbreviated St, cf. Fig. 1. TMA (tetramethylammonium), lobeline, DMPP (I. I-dimethyl-phenylpiperazine), and PTMA (phenyltrimethylammonmm) also have nicotine-like effects but weaker than those of nicotine and decreasing m the order they are listed, cf. the concentration dependent reactions in Table IA, a and Figs 2c, d. Common to all the nicotinic agents is that their effects are quickly reversible in pure sea water which indicates that they are mediated by superficial receptors. A very strong agonist-receptor interaction evidently leads to intense, tetanic C-activity, quickly followed by C-paralysis as well as by ciliary paralysis,
apparently a srgn of overstimulation~ a weaker stimulation, on the other hand, may provoke prolonged C-activity without ciliary paralysis. This complicates the dose-response curves. In addition, these curves are shifted at low salinity, i.e. the tetanic activity is replaced by an extended period of strong C-activity and the C-paralysis IS gradual and incomplete. cf. Fig. 3 where the late, weak C-activity represents movements with very low amplitude, a state close to paralysis. A similar reaction occurs at normal salinity if the larvae are very young, somewhat less than 3 days, presumably a sign of some immaturity of the signal system. The C-stimulation IS strongest in wellpigmented larvae (cf. the discussion).
The effects of agents wtth a pure or dominating muscarmic activity are summarized in Table IA. b which shows that the C-activity, in most cases, remains at a low level and this within a range of concentrattons from around 4 x 10~4-10~b M (cf. Fig. 4a-c). The state of the cilia is not affected Furthermore, spontaneously unquiet larvae are calmed down. At the higher concentrations within this range, a certain elevation of the S-activity can be seen. This and other observations suggests that the S-activity is normally hampered by the C-signals (cf. the discussion). This picture is applicable to muscarine, oxotremorine. bethanechol, metacholine, and also to pilocarpine and arecoline but in these cases only from IO-” M downwards. That the muscarinic effect of pilocarpine and, m particular, arecoline appears to be combined with a pronounced nicotimc action is seen at very high concentrations, e.g. 4 x 10W4M, where an initial C-peak precedes an almost compiete C-paralysis followed by new C-peaks or a progressive C-activation (Fig. 4d). Like the effect of nicotine, the increased activity is quickly reversible in sea water, cf. the slow reversibility of the pure muscarinic effects mentioned
Muscular
activity
in the sea urchin.
I.
3
Table IA and B Effects of nicotituc and muscarinic agents and agents wtth a mtxed effect mcludmg chohnesterase inhibitors on the C-activity and the state of the ammal ctlia. Act : x symbols refer to the degree of stimulation, nntial respective delayed and of longer duration: 0 mdtcates no or very weak stimulation. Tet: x symbols refer to initial tetanic C-activity; 0 mdicates lack of tetamc activity although a marked initial C-peak may occur. Paralys.: x-symbols refer to the occurrence of complete respectively incomplete C-paralysts; 0 indtcates lack of C-paralysis. Late act.: x-symbols refer to the late C-activity, e.g. after C-reactivation, O-symbols refer to prolonged C-paralyses. Cil. paralys : x-symbols refer to prolonged ciliary paralysis; O-symbols Indicate lack of ctliary paralyses although the ciha may show some stgns of tmtation; x to 0 Indicates temporary ciliary paralysis until C-reactivation occurs Agents
Act.
(a) Ntco. IO-P-10-s 10-6
xx x to xx
TMA 10 -’ 10-J 10-e
Paralys.
Tetany
:
Late act. 0 xx
:
xx
X
t :;
c) 0
;r 0 0 0
Cil. paralys.
;,
0 xx 0
X
X
t”x,
0 0 0
x to 0 0
DMPP 10-s 10-4 10-b
;x;
0 0 0
PTMA 10-1 10-4 10-6
t:; 0
0 0 0
0 0 0
;x, 0
0 0 0
0
0
0
0
0
xx
0
(0)
xx
0
xx
tb) Mow.* 4 X 10 4-10-6 Polo and areco 4 x 10 ’
‘Must. refers to muscarme, oxotremorme, bethanechol, metachohne and also to ptlocarpme and arecohne from 10~4 M downwards Agents (a) Carba. 10-s 4 X 10-q 10 -4 10-b ACh 10-s 4 X 10-4 10 s 10-b (b) Physo. 10~~4 10-s 10-e 10-1 10--s
Act. xx xx
Tetany.
Paralys.
X
X
0
x
X
X
;,
;,
: 0 0
tt”x,, 0 0
XX XX
xx xx : xx xx xx
Late act.
xx
X
x x
x
X
Brief
0 xx
Late Dechne Dechne High P Decline Dechne
: xx x Late xxx Tetamc xxx Tetamc 0
Cil paralys x x (to 0) x to 0 0
K$ 0
x to 0 x (to 0) 0 0 0
0
:,
;I 0 0
BW 284 C 51 ]0~~4-]0-6 10-7
0 x too
0 x too
0 0
0 0
0 0
Mipafox 10-4-10~s 10-6 10-s
0 (x1 x to 0
0 0 0
0 0 0
0 t(x)) 0
0 0 0
XX
in a later section. Arecoline also affects the cilia, although complete paralysis does not occur. Agents with combined strong nicotinic and muscarinic activity Carbachol has a nicotine-like effect. There are, however, some important reservations to this, cf. Table lB, a (also Fig. 9 in Gustafson and Treufeldt, 1981). The C-paralysis which follows the initial tetanic burst may indeed be permanent or of very long duration, but only at concentrations down to 4 x 10e4 M, and C-paralysis for more than 15 min only occurs down to lo-’ M. Furthermore, contrary to the case with C-paralyzing nicotine solutions, the
to 0
C-paralysis is attended by very pronounced S- and L-activity. In addition, once the C-movements reappear, they are in general extremely frequent and of high amplitude and foreshadowed by the closely related P-movements. C-paralysis is attended by ciliary paralysis, but the cilia are reactivated in connection with the C-reactivation. The muscular reactions suggest that the strong nicotine-like action of carbachol is moderated by its muscarinic effect. This is in line with the response to carbachol 10e6 (cf. Fig. 9 in Gustafson and Treufeldt, 1981). In this case the strong C-activation declines but without a parallel decline in the amplitude of the C-movements, i.e. contrary to the low salinity
TRYGGVECUSTAFSON
Fig. 2. Effects of mcotinic agents at normal salinity. (a) Nicotine IO-’ M (b) Nicotine 10m6M (c) TMA 10~’ M (d) DMPP 10 -4 M Y-axls. Frequency of movements; X-axls: Time m hrs; for further definitions see section “Diagrams”. C-activity indicated by round dots, S-activity by f signs, L-activity by rhombic dots, C-curve also marked by a C. T indicates tetanic C-activity. response to nicotine 10 -’ in Fig. 3, I.e. the decline apparently not due to overstimulation but to muscarimc calming down. The muscarinic action carbachol may also be responsible for the lack prolonged C-paralysis at very high concentrations
IS a of of of
Fig. 3. Effects of nicotine 10e4 M at a reduced salinity, 21 promille. Y- and X-axis and symbols for C-. S-, and L-activity defined m legend to Fig. 2.
carbachol after premcubatlon of the larvae with low concentration of the same agent (cf. Figs 4 and 8 in Gustafson and Treufeldt, 1981). Such an “adaptive” effect has so far never been observed in similar experiments with nicotine. The spontaneous C-reactivation after carbachol paralysis may also reflect a muscarine-induced “adaptation” (cf. Fig. 9 in Gustafson and Treufeldt, 1981 and the effect of strong pilocarpine and arecoline). Stimulation and C-paralysis is quickly reversible in sea water and repeated exposure to carbachol brings about renewed paralysis provided the previous exposure was not too long (cf. Fig. 7 in Gustafson and Treufeldt, 1981). As in the experiments with nicotine, the dose-response relations depend on the salimty. At 21 promille, the effect of carbachol4 x 10s4 M is similar to that of carbachol 10m6M at high salinity whereas carbachol 10e6 M is only slightly stimulatory. The reactions are the same in very young larvae. The C-stimulation is always strongest in well pigmented larvae. In experiments with acetylcholme, fresh solutions were frequently applied during the course of the recording. The effects are summarized in Table 1B, a. One may suggest that the nicotinic effect of acetylcholine is strongly moderated by its muscarinic action as long C-paralysis only occurs at a very high concentration, 10e3 M. In addition, this paralysis is attended by high P-activity, a reaction closely related to the C-movements (Fig. 5a). At 4 x 10m4 M there is at most an extremely brief C-paralysis. The C-activity rises to very high values after which it declines to a
Muscular
activity
in the sea urchin. 1. (d)
Fig. 4. Effects of muscarinic agents. (a) Muscarine Philocarpine 10e4 M (d) Arecohne 5 x 10m4 M. Transfer symbols for C-. S-, and L-activity
moderate level, presumably due to a progressive muscarinic action since the amplitude of the C-movements is unchanged (Fig. Sb). At lo-’ M and lower concentrations there is at most a very brief activation followed by a muscarine-like calming down (Fig. 5~). The vigorous stimulatory effect of acetylchohne 1O-4 M is strongly counteracted if the larvae are pretreated with low concentrations of this agent. One may assume also that this “adaptation” reflects the muscarinic effect during pretreatment. Ciliar paralysis only occurs in connection with C-paralysis. The C-stimulation is reduced at very low salinity but is favoured by high pigmentation. The effects of proprionylcholine and buturylchohne are similar to that of acetylcholine. Cholinesterase
inhibitors
The effects of three cholinesterase inhibitors, physostigmine, mipafox, and BW 284 C 51 (benzyldimethylguanidine, Wellcome Research Laboratories) have also been investigated. The action of physostigmine was described in an earlier report (cf. Fig. 3 in Treufeldt and Gustafson, 1981), but after this some further data have been collected (cf. Table 1B, b). At 10e9 M the effect is a clear muscarinic one, as at low concentrations of acetylchohne, but already at concentrations around IO-’ M there is a vigorous, nicotine-like C-activation, gradually characterized by intermittent tetanic periods at the brink of paralysis but never going beyond it, presumably saved from this by the indirect muscarinic activity. At lo-‘M, however, the effects are similar to those of strong nicotine and carbachol. The results thus suggest that physostigmine, apart from its action as a
2 x 10e4 M (b) Oxotremorine 5 x 10e5 M (c) to pure sea water at arrow B. Y- and X-axls and defined in legend to Fig. 2.
chohnesterase inhibitor, has a direct nicotine-like effect on a superficial receptor (cf. a later section). That the stimulation as well as the paralysis is quickly reversible upon early interruption of the treatment is in line with this assumption. The C-paralysis by physostigmine is followed by strong reactivation and reappearance of ciliar activity. This change is considerably quicker at 1O-4 than at lo-’ M which suggests that the muscarinic effect of the accumulation of acetylcholine moderates the nicotine-like action. That the action of physostigmine after 1 hr of treatment at 10m4 M is only slowly reversible in sea water no doubt reflects a sustained acetylcholine effect (cf. Fig. 21 in Gustafson et al.. 1972b). At low salinity, the paralysis induced by physostigmine 10e5 M IS replaced by a vigorous peak in C-activity as in similar experiments with strong carbachol. The effects of the two other cholinesterase mhibitors, BW 284 C 51 and mipafox, differ markedly from that of physostigmine (cf. Table IB, b). This suggests that they are entirely due to the accumulation of acetylcholine, the nicotine-like effect of which is moderated by its muscarinic activity. A rather brief, initial stimulation has been recorded after exposure to BW 284 C 51 lo-‘M, at least at the onset with some tetanic tendency, particularly pronounced at high salinity (Fig. 6a). At higher concentrations, the C-activity is comparatively low, although a brief initial peak has once been recorded (Frg. 6b). Unquiet larvae are calmed down. In spite of the low stimulatory activity that dominates the picture, it is evident that the larvae are steadily affected as the C-movements occur in swarms or even slight bursts separated by calm periods. The ciliary state is unaffected, even
TRYGGVE GIJSTAFSON (a)
b)
Fig. 5. Effects of acetylcholine. (a) lo-’ M; note the P-activity, curve marked P. (b) 4 x 1O-4 M (c) 10e5 M 2 hr Y- and X-axis and symbols for C-. S-, and L-activity defined in legend to Fig. 2.
at the peaks in activity. The effects of mipafox within the concentration range 5 x 1O-4-lO-8 M reminds of those of BW 284 C 51, although no tetanic tendency has been observed.
Fig. 6. Effects of BW 284 C 51. (a) IO-’ M (b) 10m5 M Y- and X-axis and symbols for C-, S-, and L-activity defined in legend to Fig. 2. T indicates tetanic C-activity.
Nicotinic antagonists In order to shed some further light on the relationship between the nicotinic action of an agent and its C-stimulating respective C-paralysing ability. the effects of agents that block or inactivate the nicotinic receptor were studied, i.e. those of conventional nicotinic antagonists (Table 2), two snake toxins, and DTT (dithiothreitol), an agent that reversibly inactivates the nicotinic receptor (cf. Karlin et al., 1973). Most of the experiments were restricted to the concentrations used to counteract the nicotinic effects (see a later section). The results of the experiments with a long series of quaternary nicotinic antagonists at high concentrations, 10-4-10-3 M, are very consistent and may be illustrated by Figs 14 and 15 in Gustafson et al. (1972b) referring to succinylcholine 5 x 10e4 and decamethonium 10e4 M and to the lower curve in Fig. 7 in the present paper. An initial, brief and weak stimulation may occur here, followed by low activity. Spontaneously unquiet larvae are calmed down and the cilia remain active. At very low concentration, around 10m8 and lo-‘M, the stimulatory
Muscular Table
2. Effects
physostlgmme
of antmlcotmlc as revealed
actlvlty agents
in
(Ant!
by the quickness
the
sea
urchin.
1
) on the response of C-reactwatlon
to mcotine,
carbachol.
and the followmg
and
mtenslty
of
the C-movements 10m4
Nm Agents
Carba
+Antl
Decamethomum
2x
Succmylchohne
4 x 10~4
Carba
+Antl.
10~4
xx
IO
4 xxx
10-J
x
IO
J XIX
5x10 10-4
5x10 5x10
Gallamme
IO
5x10
4 xxx 4 xx
a xx
Arfonad” 10-J
(X)
x
5 x 10-J
xx
5 x 10-4
xx
5x10 IO
“Trlmetaphan
x. xx. and TXX mdvzate cle.u, column
IS based
C-paralysis counteractmn +
mdlcates
sumlar
strong
admrxed
and very strong wth
effect Induced
effect
to those of the other
of varymg
antnncotmlc
effect of decamethonium is considerable, however. but quickly fades upon increase in concentration. A similar nicotine-like stimulation has been observed with low concentrations of succinylcholine. another depolarizing antimcotme. The effects of the tertiary antinicotines pempidine. mecamylamine, and benactyzine at 10-5-10-“M are similar to those of the quaternary agents. The effect of tubocurarine is a clear exception to the general rule. At 10e4 M there is generally an enormous activation, gradually dominated by Cmovements (Fig. 7, upper curve). In fact. it IS similar to that of serotonin (cf. Gustafson et al., 1972a). It is quickly reversible in sea water. A similar stimulatory effect but of shorter duration has been recorded at high concentrations of arfonad and, in a single case, also with decamethomum. When a strong C-activation occurs, It 1s always attended by the appearance of granules in the esophagus that swell and collect in the stomach. When such granules do not appear, the effects of the agents concerned follow the normal antinicotinic pattern. a-Bungarotoxin lo-‘M has a certain stimulatory effect. in particular to the S-activity. At IO-’ M a brief initial C-peak has been seen. The effect of cc-najatoxin within the same concentration range is
ok,,,,,,,,,,,,,,,,,,,,,,, I
2
3
d
I+
10-d
I+
5 x 10-4
I+
IO_’
xxx
1+
2 x 10~4
I+
4 xx
5 x IO_’
1-t
4 XT
5x
10
5x
10-h
antlparalyuc
effect
strategy
(antrmcotme
a varymg
to the physostlgmme
of the stlmulatory an antlpardlytlc
I+
5 x IO_’
5 1-t I+
camsylate
on expenments
or dwxtly
+ AntI.
5 x 10-J
xx
5 x 10-J
Pempdme Mecamylamme “Tetraethylammonlum.
xxx 4 xxx
10-4
Alloferm Myrolon
Phvso
(xx IO_’
Hexamethonlum TEA” Chlorrsondamme
-
do. + Ant!.
5
Fig. 7. Effects of decamethonium and tubocurarine. Upper curve: C-activity induced by tubocurarme 10m4M. Lower curve: C-activity Induced by decamethonium 10m4 M. Y- and X-axls defined m legend to Fig. 2.
solutmn)
I (Inhlbltmn)
by low concentrations mtenslty
The physostlgmme
The
admlxed
after
symbolizes
of physostlgmme.
effects of tubocurarme
1s
agents
similar as is DTT 10-j and 2 x 10m3 M. The reaction between the agents concerned and the nicotinic receptor thus appears to bring about a certain, more or less transient. activation. The state of the cilia at the animal plate. however, IS unaffected. Attenuation agents
qf the nicotinic
effects
by antinicotinic
As most of the antinicotinic agents treated in the preceding section do not have any pronounced or at least no persistent stimulatory effects at appropriate concentrations, they could be used as tools in a further analysis of the nicotinic effects. The experiments were carried out with varying strategy (cf. Table 2). As certain antinicotinic agents may have side effects, even muscarinic ones, it was important to use a wide variety of these compounds. However, their effects on the response to nicotine-active agents are quite consistent (cf. the Table). Upon simultaneous exposure to nicotine 10m4 M and nicotine antagonists, the C- and ciliary paralysis is counteracted, instantaneously or with short delay (cf. Fig. Sa, b). When the C-reactivation is delayed. it is preceded by marked S- or L-activity or both. The C-reactivation also occurs if the antagonists are added to larvae already paralyzed by pure nicotine. The effect is weaker, however, which suggests that the paralytic effect of nicotine is cumulative. In experiments with carbachol 4 x 10m4M, the results are analogous, although the C-reactivation is often preceded by marked P-activity. After pretreatment with antagonists before the admixture of carbachol, the paralysis fails completely. In experiments with decamethonium, a concentration of lo-’ M is enough to reactivate the C-movements of larvae in the presence of carbachol lo-’ M, instantaneously and very strongly. In fact, the most powerful effects are always seen when the concentration of the antagonist is below that of carbachol (cf. Fig. 9). A prerequisite for high C-activity is evidently that the remaining nicotinic effect is rather high. The strong decline in C-activity, without decrease in the amplitude of the C-movements, that
TRYGGVE GUSTAF~ON
Fig. 8. Effects of antimcotinic agents on the response to mcotine and carbachol in C-paralyzmg concentrations. (a) Decamethonium 10e4 M + nicotine lo-’ M (b) Succinylchohne 10mJ M + nicotme 10m4 M (c) Effects of pure carbachol 4 x lo-” M, added at the arrow after premcubation with pure succinylcholine lo-) M for 1.25 hr showing quick reversibility of the antinrcotinic effect; some P-activity IS shown in the curve marked P. Y- and X-axis and symbols for C-, S-. and L-activity defined m legend to Fig. 2. Curve indicated by triangles and P refers to P-activity. T indicates tetanic C-actlrity
occurs after a period of reactivation, may be due to the muscarinic effect of carbachol. The effects of the antinicotmes are quickly reversible. The reversibility may lead to complete paralysis m pure carbachol (Fig. 8~). However, in larvae which have been subjected to prolonged treatment with a mixture of carbachol and an antinicotine, paralysis fails upon exposure to pure carbachol, supposedly due to the muscarinic effects of carbachol during the preceding exposure (cf. an earlier section). The muscarinic effects will be further elucidated in a later section. a-Bungarotoxin 6 x lo-‘M counteracts the Cparalysis of admixed carbachol 4 x IO-“ M (Fig. 10a). The toxin effect is also distinct at lo-‘M, although the C-reactivation is replaced by the P-movements forerunners of C-activity, i.e. (Fig. lob). The cihary paralysis is also counteracted to a varying extent. A clear antiparalytic effect re-
mains after transfer to pure carbachol. The antiparalytic effect of a-najatoxin is presumably weaker than that of a-bungarotoxin, at least it is more quickly reversible after transfer to pure carbachol, although the antiparalytic effect on the cilia persists. DTT 10m3 M also prevents the C-paralysis and to some extent also cihary paralysis. The protective effect is almost completely reversible in pure carbachol. That physostigmine, apart from its action as a cholinesterase inhibitor, appears to have a direct nicotine-like effect is supported by numerous experiments (cf. Table 2). A long series of antinicotines thus counteract the stimulatory effect of low concentrations of physostigmine, its paralytic effects at lo-’ M (cf. Fig. II), and also the high activity of larvae spontaneously C-reactivated in physosttgmine 1O-4 M. The C-paralyzing effect of nicotine, carbachol, and physostigmine is not counteracted if the agents act
Muscular activity in the sea urchin. I.
Fig. 10. Effects of ~-bungarotoxin on the response to a paralyzing concentration of carbachol; P-activity is shown
Frg. 9. Effects of decamethonium on the response to a normally C-paralyzing concentration of carbachol; diagram restricted to the C-activities; curve 1: De~methonium 2 x 10e4 M + carbachol 4 x 10e4 M, curve 2: Decamethonium 4 x lo-“M +carbachol 4 x 10e4M. Y- and X-axis defined in legend to Fig. 2.
together at high concentrations, but lobeline and PTMA decrease the paralytic effect of nicotine. It thus appears that less effective nicotine agonists have a certain capability to displace nicotine from its receptor. Attenuation of the nicotinic effect by muscarinic agents In some previous sections, it was suggested that the nicotinic effects of agents such as carbachol, acetylcholine, and physostigmine are moderated by the, direct or indirect, muscarinic effects. One may therefore expect that the paralytic effects of nicotine would also be weakened or turned into strong stimulation in the presence of pure muscarinic agents, acetylcholine, BW 284 C 51, mipafox, respectively very low concentrations of physostigmine, and that such agents would further reduce the paralytic effect of carbachol and physostigmine. The results of a great number of experiments are in line with this expectation (cf. Table 3 and the examples in Fig. 12ax). They also show another prominent regularity that is quite illuminating (cf. the following). The maxima1 C-activity upon exposure to a mixture of nicotine and agents with a pure or dominating muscarinic activity depends on their proportions (see Table 3 and Fig. 12b and curve 2 in d). It is much higher at rather low concentrations of the muscarineactive agents concerned than at higher concentrations. It is therefore clear that high muscular activity is favoured by a proper balance between the nicotinic and the muscarinic signals. A similar conclusion can
curves P. (a) cc-bungarotoxin in the marked 6 x lo-’ M +carbachol 4 x 10e4 M (b) a-bungarotoxin IO-’ M + carbachol 4 x 10S4M after 5.5 hr pretreatment with pure toxin solution. Y- and X-axis and symbols for C-. S-, and L-activity defined in legend to Fig. 2.
be drawn from the experiments with nicotine or carbachol plus cholinesterase inhibitors (cf. curve 1 in Fig. 12d), but the nicotinic contribution of the acetylcholine piled up should not be disregarded. The role of the concentration relationships is also illustrated in Fig. 12b where the temporal changes in the C-activity is plotted in two nicotin~oxotremorine experiments. It is evident that the C-activity drops when it has reached a maximum (cf. also the decline in Fig. 12a), and that the decline is accentuated when the oxotremorine concentration is increased from lOas to low4 M as in Fig. 12b. One may compare this with the effects in Fig. 9 which in fact also illustrate the activities at varying nicotinic and muscarinic provocation. However, in experiments with stronger nicotine concentration, 2 x 10M4M, a high C-activity is reached also at very high concentration of oxotremorine, 4 x 10e4 M, but here also there is a sharp decline (Fig. 12~). The antiparalytic effect of muscarinic agents and the characteristic time-course of the C-activity is also
Fig. 11. Effect of alloferin 2 x 10m4M on the effects of physostigmine at stimulatory and paralyzing concentrations. Pretreatment with pure alloferm for 45min; physostigmine 5 x IO-‘M admixed at first arrow; physostigmine 10m5M admixed at second arrow. Y- and X-axis and symbols for C-, S-, and L-activity defined in legend to Fig. 2.
-9
-8
-7
-6
-5
-4
Fig. 12. Effects of muscarmic agents mcluding mipafox on the response to nicotine-acttve agents at C-paralyzing concentrations. (a) Oxotremorine 4 x IO-” M -I-carbachoi 4 x tom4 M (b) 1. C-activity at oxotremorine lo-’ M c nicotme 10e4 M; oxotremorine concentration increased to 10m4M at the thick arrow; 2. C-acttvity at oxotremorine low4 M + mcotine IO-” M. T-symbol for tetanic C-activtty only refers to the upper curve (c) Oxotremorine 4 x lO-4 M fnicotine 2 x lo-” M (d) The maxtmal C-activity reached upon exposure to C-paralytic nicotinic agents plus muscarinic agents in molar concentrations indrcated by exponents at the abscissa. Curve 1. Mipafox fcarbachol 4 x 10e4 M; curve 2. Oxotremorme + nicotine 10e4 M (e) Slow reversal of the betanechol effect after exposure to betanechol 6 x 10m4M for 2.5 hr; pure carbachol 4 x 10-4M added at the arrow. Y- and A’-axis and symbols for C-, S-, and L-actrvtty (in diagrams a-c and e) defined in legend to Fig. 2 T Indicates tetamc C-activity.
Muscular activity In the sea urchin. I. evident in experiments where physostigmine 10e5 M is combined with strong betanechol. The powerful stimulation brought about by low, non-paralyzing concentrations of physostigmine also decreases upon admixture of betanechol and also the spontaneous C-reactivation level in physostigmine 10m4M. The muscarinic effects in these experiments instantaneously reversible (cf. Fig. 12e).
are not
Antlmuscarines
As the muscarmes in the results reported counteract the effects of mcotinic agents, it may be supposed that a nicotine-muscarine interaction also plays a role in the intact larva, and that the muscarinic function serves as a “brake” that counteracts excessive activity. If so, one could expect that antimus-
carinic agents may release a high muscular activity. The effects of a series of antimuscarines at high concentrations are in line with this assumption. Atropirie at concentrations down to at least 5 x 10e5 M thus bring about a very strong C-activation, often with some delay. A clear stimulatory effect has also been recorded with scopolamine 5 x 1O-6 M. At low salinity the effect consists of a long series of peaks which, in the long run, tend to decrease in height as well as in their duration (Fig. 13a). At high salinity the peaks fuse into a block followed by a strong decline in activity or even total paralysis (Fig. 13b). The effects of atropine and scopolamine are reproduced by methylatropine and oxiphencyclimine. The reversibility of the antimuscarine effect is quick, except when total paralysis has been reached.
3%.
300.
11
(b)
2s
zoo 150
loo
50
0
(d) ic
Fig. 13. Effects of atropine and its counteraction by a muscarinic agent. (a) Atropme lOa M at a sahnity of 21 promille (b) Atropine 10m4M at normal salinity (c) Atropine lOA M + betanechol 10e4 M after pretreatment with betanechol 10m4M for 2.5 hr; atropine admixed at the arrow. Y- and X-axis and symbols for C-, S-, and L-activity defined in legend to Fig. 2. T indicates tetanic C-activity.
TRYGGVEGUSTAFWN
12
(b)
iic
.
I i
I l
. Pi
l \
\.
Fig. 14. Effects of atropme on the response to mcotine and physostigmme. (a) Atropine 5 x 10m4 M + nicotine 10m4 M (b) Atropine 10m4 M + physostigmine 10m5 M; note the scale. Y- and X-axis and symbols for C-. S-. and L-activity defined in legend to Fig. 2. T indicates tetanic C-actwity
This indicated that the agents interfere at a superficial level. That their effect is counteracted by oxotremorine. betanechol, and by low concentrations of BW 284 C 51 and physostigmine may suggest that muscarinic receptors are involved (cf. Fig. 13~). That the antimuscarines may have other effects as well should not be neglected, however (see the following). If the antimuscarines only weaken the muscarinic “brake”. the admixture of nicotine low4 M would easily lead to overstimulation followed by C-paralysis. This is partly in line with the results of the experiments carried out (cf. Fig. 14a). The further course of the activity curve, however, suggests that atropine at high concentrations has a certain antinicotinic effect as some C-reactivation occurs. With isopropamide. another antimuscarine. the antinicotinic effect is considerably stronger. The paralytic effect of carbachol 4 x 10e4 M is also effectively counteracted by the whole series of antimuscarines. An unprecendently intense stimulation of long duration is triggered in experiments with atropine 10m4 M plus physostigmine 10e5 M (cf. Fig. 14b, note the scale). One may think that the paralyzing nicotine-like effect is here decreased into a preparalytic and therefore strongly stimulatory intensity due to the antinicotinic effect of atropine which, at the same time, retards the muscarinic calming down. DISCUSSION
The pattern of contractile activities in the intact sea urchin pluteus is rather regular and monotonous (cf. Fig. 4 in Treufeldt and Gustafson, 1981). One may therefore assume that a pacemaker-function is involved. Indeed, there may be two different pacemakers, one with a comparatively high basal frequency and only responsible for the muscular
activity, one with a low frequency (around 8 impulses per hr) controlling the muscular as well as the intestinal movements-the stomach and proctodaum cycles (cf. Fig. 1 in the present paper and Figs 6. 8. and 9 in Gustafson et al., 1972a). Both pacemakers may be further activated and paralyzed in a similar way, although their relative role may vary in the course of an experiment (cf. Treufeldt and Gustafson. 1981). An interpretation of the organization of the pacemaker-system is presented in Fig. 5 in the report just quoted. The results so far reported indicate that the nicotine-active agents are stimulatory and that Cparalysis as well as ciliary paralysis, that immediately follows a vigorous, tetanic, peak in C-activity, IS the result of excessive stimulation. Agents wtth pure muscarinic activity, on the other hand, keep the muscular activity low, calm down spontaneously unquiet larvae, and do not affect the ciliary state. The rapid effects of the agents concerned and their antagonists indicate that they interfere with superficial receptors involved in the muscular as well as in the ciliary control. The quick reversibility of the nicotmic agents is in line with this. The delayed reversibility of the muscarinic effects will be discussed later. As the dose-response relation of purely nicotinic agents as well as that of carbachol is changed at lowered salinity, one may assume that the nicotinic effects are mediated by an increased influx of some ions, probably Ca’+ (see a later report in this series). The muscular and ciliar paralysis may therefore reflect an excessive ionic influx. As the muscarinic effects on unquiet larvae may be mimicked by reducing the salinity, it is logical to conclude that the muscarines decrease this influx (see a forthcoming report). Due to this action they may counteract nicotinic stimulation and paralysis.
Muscular activity in the sea urchin. I. That the effect of carbachol and, in particular, that of acetylcholine differs from that of nicotine is in line with the fact that these agents, in many systems, have nicotinic as well as muscarinic effects. In the sea urchin pluteus, the muscarinic effect of acetylcholine is apparently more strongly expressed than that of carbachol. However, the muscarinic effect of carbachol is clearly manifested when antinicotines are admixed (Fig. 9), cf. also the decline in C-activity without decrease in C-amplitude at low salinity. It has already been suggested that the “adaptive” effect during exposure to carbachol, including the spontaneous C-reactivation, reflects a muscarinic action. A general impression is that the threshold for the muscarinic effect in the pluteus larva is much lower than that of the nicotmic agents. In line with this, the effects of low concentrations of acetylcholine are predominantly muscarinic. When agents with pure nicotinic and muscarinic activity are combined, the ratio between the two effects is of decisive importance, both for the maximal C-activity reached after paralysis and for the further course of the activity curve. The role of this ratio is also illustrated in experiments with carbachol-treated larvae exposed to varying concentrations of antinicotines (Fig. 9). The effects of the cholinesterase inhibitors BW 284 C 51 and mipafox are evidently mediated by acetylcholine accumulation. Its nicotinic stimulation is here hampered by its muscarinic effects. The action of physostigmine, on the other hand, suggests that this agent also has marked, direct nicotinic effects. This is in line with the effects of antinicotines on physostigmine-treated larvae. In the long run, which is particularly clear at high concentrations, its nicotinic effect is moderated by the acetylcholine accumulated. The antiparalytic effect of all the cholinesterase inhibitors at low concentrations are evidently due to the indirect muscarinic effect. The results m Fig. 12e and a series of similar experiments indicate that the muscarinic effects, e.g. on the guanylate cyclase system, are not immediately reversible. One may ask if this reflects an after-effect on this system. It may be added that the muscarinic effect of oxotremorme is strongest at high salinity, an observation that might reflect the importance of Ca’+-ions in the activation of the cyclase. The C-activity in a larva paralyzed by TMA sooner or later reappears, in spite of the lack of direct muscarinic activity (Fig. 2~). This could depend on acetylcholine released from an inner source (see the end of the discussion). However, if the excitation is mediated by an influx of Ca’+-ions, this may also lead to an activation of the guanylate cyclase system, a presumed target for the muscarinic action. Such a mechanism. however, is apparently insufficient to reactivate larva paralyzed by very strong nicotinic treatment. The role of the guanylate cyclase system will be further discussed in a forthcoming paper in this series. That the activity of an intact larva is governed by a certain muscarinic “tonus” is suggested by the strong stimulatory effects of antimuscarines at high concentrations, an effect which is counteracted by muscarines. One may say that this “tonus” serves as a “brake”. However, the antimuscarinic action appears to be contaminated by an antinicotinic effect. It
13
may even reflect direct effects on some ionic channels. The different muscular elements are all derived from the celomic rudiment (cf. Fig. 1 in this paper and Gustafson and Wolpert. 1963). One may therefore ask how one type of muscular activity may dominate at the expense of the others, and why it even may be monopolized. A preliminary working hypothesis is that the different muscular elements have different thresholds of excitation. The C-movements have a relative long duration and are also more forceful than the twitch-like L-movements, sometimes even tetanic. They may therefore require very powerful signals. One may also suggest that these signals somehow suppress the signals to the other muscular activities, cf. the S-favouring effect of the muscarines that decrease the C-activity and the opposite effects of serotonin and some catecholamines described in Gustafson er al. (1972a) and later reports in this series. In addition, a very high L-activity may make the peristaltic system incapable of producing the concerted, consecutive L-twitches involved in the peristaltic waves denoted by S-movements, cf. heartflimmer. The C-stimulatory effect of nicotinic agents is particularly strong in pigment-rich larvae. The relationship between the pigment formation and the monoamine metabolism demonstrated by Asashima (1972a, b) may therefore indicate that the C-stimulation due to an increased ionic influx, e.g. induced by nicotine, is mediated or amplified by an inner monoaminergic system. The main anatomical localization of this system is, hypothetically, the mouth region (cf. Fig. 5 in Treufeldt and Gustafson, 1981). a site that, according to histochemical investigations, does contain monoamines (cf. Fig. 5 in Ryberg, 1974). Monoamines released from this system may also be responsible for the suppression of the S-activity, cf. the effects of serotonin and various catecholamines (Gustafson et uI., 1972a) and later papers in this series. That the release may be governed by acetylcholine is in line with the observation that the monoamine containing structure is closely related to, or possibly identical with, high specific cholinesterase activity (cf. Fig. 26c in Gustafson et al., 1972b). The aberrant monoamine-like effect of tubocurarine etc., which is correlated with the release of some granules into the esophagus, may be related to a massive release of monoamines from the site just discussed. In any case there are metachromatically staining cells in its vicinity that may be the source of the granular material that appears in connection with strong C-stimulation by these agents (cf. Ryberg and Lundgren, 1975). That tubocurarine and some other antinicotinic agents at high concentration may release pharmacologically very active agents is well known indeed (cf. Goodman Gilman et al., 1975). The signal system controlling muscular activity in the pluteus is apparently closely related to the one controlling ciliary activity in the embryo (cf. Soliman, 1983a, b, 1984a, b). An important question is how the embryonic system develops pacemakers with a comparatively slow rhythm. Closely connected with this question is how strong a nicotinic stimulation may turn off the pacemaker-signals eliciting C-activity. One may speculate that the calm periods between the
14
TRYGGVE GUSTAFSON
pacemaker-bursts and the long paralysis after strong nicotinic stimulation reflect the same mechanism, e.g. a hyperpolarizing outflux of K+-ions induced by the influx of Ca2+ (cf. Meech, 1980). This question will be further discussed in later reports in this series. In conclusion, the muscular and cihary activity in the sea urchin pluteus is affected by nicotinic as well as muscarinic agents. The nicotinic effects are stimulatory, but excessive stimulation is quickly followed by paralysis, in particular in the most powerful muscular strands. Muscarinic agents, on the other hand, have a calming effect and counteract the action of nicotinic agents. Acetylcholine, which has nicotinic as well as muscarinic effects and which occurs in the larva. is presumably involved in the control of activity of the normal larva. The cholinergic system within the larva can evidently be affected via a receptor system at the larval surface. It is suggested that the cholinergic signals bring about a release of monoamines that amplify the signals to the powerful C-activity and at the same time suppress the other muscular activities. It is also suggested that the paralytic effect of strong mcotinic treatment refect a hyperpolarizing outflux of K+ induced by a strong influx of Ca’+ which may play a central role as a charge-carrying ion. Acknorcledgements-This work has been supported by the Swedish Natural Science Foundation. I am indebted to the head of the Kristineberg Marine Biological Statton, Professor J.-O. Stromberg, and to hts staff for their never fatling generosity. I also express many thanks to Rein Treufeldt for hts patient assistance in a great number of expertments and also to docent Jan Nedergaard for his constructtve crttictsm during the preparation of the manuscript.
REFERENCES Asashtma M. (1971a) Some observations on the btosynthesis of echinochrome in the sea urchin embryos. J Fur. Ser. Clnic. Tokyo, IV. 12, 269-277. Asashtma M. (1971b) On tyrosme hydroxylase and tyrosinase activities in developmg sea urchm embryos, wtth special reference to the biosynthesis of echinochrome. J. Fat. Sri. Umc. Tokyo. IV. 12, 279-284. Augustinsson K.-B. and Gustafson T. (1949) Cholinesterase m developing sea urchin eggs. J. cell. camp PhJ~rol. 34, 311-321. Buzmkov G. A.. Chudakova I V. and Zvesdma N. D. (1964) The role of neurohumours m early embryogenesis I. Serotonm content of developing embryos of the sea urchm and loach. J. Embryol. exp. Morph. 12, 563-573. Buzmkov G. A., Chudakova I. V.. Berdysheva L. V and Vyazmina, N. M. (1968) The role of neurohumors m early embryogenesis. II. Acetylcholine and catecholamine con-
tent in developing embryos of the sea urchin. J. Embryo/ exp. Morph. 20, 119-128. Goodman Gilman A., Goodman L. S., Rail T. W. and Murad F. (1985) The pharmacological bass of therapeufits 7th edn, Macmillan, New York. Gustafson T. and Toneby M. (1971) How genes control morphogenesis. Am. Sci. 59, 452462. Gustafson T. and Treufeldt R. (198 1) Neuropharmacologtcal analysis of mechanisms controlling larval behavtour in the sea urchin. 1. Effects of carbamylcholine and acetylcholine. Acra 2001. (Stockh.) 62, 233-247. Gustafson T. and Wolpert L. (1963) Studies on the cellular basis of morphogenesis in the sea urchin embryo. Formation of the ceolom. the mouth, and the primary pore-canal. Expl Cell Res. 29, 561-582 Gustafson T., Lundgren B. and Treufeldt R. (1972a) Serotonin and contractile activity in the echinopluteus, a study of the cellular basis of larval behavtour. Evpl Cell Re.!. 72, 115-139. Gustafson T., Ryberg E. and Treufeldt R. (1972b) Acetylcholine and contractile activity m the echinopluteus. Acru Embryo1 cup. Vol 1972, 199-233 Karlin A.. Cowburn D. A. and Reuter. M. J. (1973) In “Drug Receptors”. (Edited by Rang H P.) Macmtllan, London Meech R. W. (1980) Ca”-activated K+-conductance. In Moluscan Nerve Cells From Biophystcs to Behavtour (Edited by Koester J. and Byrne J H.). pp 93-103. Cold Spring Harbour Laboratory. Ryberg E. (1974) The localizatton of btogenic ammes m the echinopluteus. Acfu Zool. (Stockh.) 55, 1799189. Ryberg E. and Lundgren B. (1975) Secretory cells m the foregut of the echmopluteus W’ilhelm Roux’ Arch. 177. 155-262 Sohman S (1983a) Pharmacological control of ciliary activity m the young sea urchin larva Effects of chohnergtc and anttcholinergic agents Comp Biochem Physrol. 74C. 397407. Sohman S. (1983b) Pharmacological control of ctliary activtty m the young sea urchin larva. Effects of monoammergtc agents. Comp. Blochem Physrol. 76C, IX1 ~191. Sohman S (1984a) Pharmacologtcal control of cthary acttvity m the young sea urchin larva. Studies on the role of Cal+ and cyc11c nucleotides. Camp Birwhem. Phpriol. 78C. 183 191. Sohman S. (1984b) Pharmacological control of ciliary acttvity in the young sea urchin larva: Chemical studies on the role of cychc nucleottdes. Camp Blochem. Phyriol 78C, 175-181. Toneby M. (1977) Determination of 5-hydroxytryptamme in early echinoderm embryos. Comp. Ciochem. Phwrol SC, 77-83 Toneby M. (1980) Dopamine m developmg larvae of the sea urchin Psammechinus mdrarls GMELIN. Camp. Biochem Ph_wol. 65C, 139-142. Treufeldt R. and Gustafson T. ( I98 1) Neuropharmacologtcal analysts of mechanisms controllmg larval behavtour m the sea urchin 2. Effects of physosttgmme and the role of cholinergtc mechanism. At/a Zool. (Srockh.) 62, 249 -258.
1989
0306-4492/89 53 00 + 0.00 Pergamon Press plc
PHARMACOLOGICAL CONTROL OF MUSCULAR ACTIVITY IN THE SEA URCHIN LARVA. I. EFFECTS NICOTINIC AND MUSCARINIC AGENTS
OF
TRYGGVEGUSTAF.WN The Wenner-Gren
Institute,
Biologihus
F3, University (Received
25 Muy
of Stockholm,
S-106 91 Stockholm,
Sweden
1988)
Abstract-l. The muscular system of the sea urchin pluteus is stimulated by nicotinic agents. Excessive stimulation is followed by paralysis of the most powerful muscular strands. The effects are counteracted by antinicotmic agents. 2. Muscarimc agents keep the activrty low. calm down initially unquiet larvae, and counteract the nicotinic effects. 3. The effects are quickly elicited and in general are also quickly reversible in sea water, whtch indicates that most of the receptors mvolved occur at the larval surface. 4. It is suggested that the nicotinic and muscarinic signals control certain ionic fluxes in opposite directions, and that the stimulatory effect on the most powerful muscular strands is amplified by a monoaminergic system.
INTRODUCTION The muscular system of the mature pluteus has been used as a model that may shed some light on the pharmacological signals which appear to control the morphogenetic movements in the embryo as well as its ciliary activity, cf. Gustafson and Toneby (1971) and Soliman (1983a, b; 1984a, b). An advantage of the system and technique used is that the reactions are quick and distinct, and that the reversibility of the effects of various agents and their interaction can be investigated. In addition, several types of contractile activity can be studied. This system may also elucidate some properties of a primitive pacemaker system. This may be particularly important as the vertebrates and the echinoderms may have a common ancestor. The anatomical organization of the system is described in Fig. 1 which also defines the activities the twitch-like L-movements, concerned, the peristaltic S-movements made up of consecutive Ltwitches, the powerful C-activity and the closely related P-movements. It also shows some changes in the ciliary system at the animal plate or velum; St indicates ciliary paralysis. The different muscular movements can be combined in various ways and appear to be pacemaker-controlled. One may say that the pacemaker-signals may choose from a menu of various activities. Biochemical and histochemical studies show that the sea urchin larva contains cholinesterases, acetylcholine, and various monoamines, cf. Augustinsson and Gustafson (1949), Buznikov et al. (1964, 1968), and Toneby (1977, 1980). The aim of the present program is to further investigate this system, not least the mechanism of action of the cholinergic and monoaminergic agents. Preliminary results on the muscular effects of a number of pharmacological agents have been reported earlier (Gustafson et al.,
1972a, b). A detailed account of the stimulatory and paralytic effects of carbachol and physostigmine has also appeared (cf. Fig. 9 in Gustafson and Treufeldt, 1981 with Fig. 3 in Treufeldt and Gustafson, 1981). In the present paper the effects of a series of cholinergica have been studied in order to elucidate to what extent their different responses are mcotinic or muscarinic or reflect a nicotinic-muscarinic interaction. The actions of catecholamines, serotonin, cyclical nucleotides, and their relation to the flux of Ca*+ and K+ will be discussed in forthcoming reports in this series. MATERIALSAND METHODS The material used is the same as in our earlier investigatrons i.e. 3 day olu plutei of Psammechinus miliaris, of both S- and the Z-form living at different depths and therefore at different salinities, 34-30 respecttvely around 25 promtlle. The technique applied was described in earlier papers (Gustafson et al., 1972a; Gustafson and Treufeldt, 1981). RESULTS The description of the results may appear tedious, in particular as the muscular system is rather intricate, and the terminology may look exotic. However, most of the facts are important for the final interpretation of the muscular control mechanism, cf. the discussion. Diagrams
The muscular effects of the agents concerned are illustrated in diagrams where the frequency of different movements, i.e. the number of movements during each of the 15 min long periods of recording (Y-axis), is plotted against time in hr after the control period (X-axis) except in Fig. 12d. Each diagram refers to a single experiment. The application of the
TRYGGVE GUSTAFS~N
Fig. 1.Anatomy of the contracttle system of a 3-day old plutcus of Psa~n~ze~~zzn~.~ mrbarzs and the variation m the state of the ctlia at the antmal plate from normal to paralyws: the L-, S-, C-, and P-nlovements are defined by arrows; upon strong stimulatton the C-movements may be tetanic (see the symbol T in the dragrams) and strong tetanic activity IS followed by paralysis of the C-movements; the N-movements first appear in 4-day old larvae; St mdlcates cihary paralysis Modtfied after Gustafson and Treufeldt (1981)
agents concerned is indicated by arrows. The C-activity is marked by round dots (Fig. 12d being an exception), the S-activity by + signs, the L-activity by rhombic dots. and the P-activity by triangles. Intense tetanic C-activity is marked by a large T; the tetanic intensity is often so strong that individual C-movements cannot be recorded. A somewhat weaker tetanic C-activity is indicated. Agents with pure nicotinic uction The effects of these agents are summarized m Table IA. a. At high concentrations of nicotine, 10 -“-IO-” M, occasionally also at 5 x IO-‘M, there is an immediate burst of strong, tetamc C-activity, instantly followed by permanent C-paralysis, There is also a strong activation of the S- and L-movements which, at the highest concentration, is followed by low activity or even by paralysis (Fig. 2a). At 2 x IO-’ M the initial C-activity is weak but followed by a strong, progressive C-activation (Fig. 2b). At lo-‘M, only a moderate stimulation may be seen The C-paralysis is accompanied by an instant change in the state of the ciha, in particular at the animal plate. The cilia thus become stiff and immobilized. a change that may be denoted “ciliary paralysis”, in earlier reports called stiffening, abbreviated St, cf. Fig. 1. TMA (tetramethylammonium), lobeline, DMPP (I. I-dimethyl-phenylpiperazine), and PTMA (phenyltrimethylammonmm) also have nicotine-like effects but weaker than those of nicotine and decreasing m the order they are listed, cf. the concentration dependent reactions in Table IA, a and Figs 2c, d. Common to all the nicotinic agents is that their effects are quickly reversible in pure sea water which indicates that they are mediated by superficial receptors. A very strong agonist-receptor interaction evidently leads to intense, tetanic C-activity, quickly followed by C-paralysis as well as by ciliary paralysis,
apparently a srgn of overstimulation~ a weaker stimulation, on the other hand, may provoke prolonged C-activity without ciliary paralysis. This complicates the dose-response curves. In addition, these curves are shifted at low salinity, i.e. the tetanic activity is replaced by an extended period of strong C-activity and the C-paralysis IS gradual and incomplete. cf. Fig. 3 where the late, weak C-activity represents movements with very low amplitude, a state close to paralysis. A similar reaction occurs at normal salinity if the larvae are very young, somewhat less than 3 days, presumably a sign of some immaturity of the signal system. The C-stimulation IS strongest in wellpigmented larvae (cf. the discussion).
The effects of agents wtth a pure or dominating muscarmic activity are summarized in Table IA. b which shows that the C-activity, in most cases, remains at a low level and this within a range of concentrattons from around 4 x 10~4-10~b M (cf. Fig. 4a-c). The state of the cilia is not affected Furthermore, spontaneously unquiet larvae are calmed down. At the higher concentrations within this range, a certain elevation of the S-activity can be seen. This and other observations suggests that the S-activity is normally hampered by the C-signals (cf. the discussion). This picture is applicable to muscarine, oxotremorine. bethanechol, metacholine, and also to pilocarpine and arecoline but in these cases only from IO-” M downwards. That the muscarinic effect of pilocarpine and, m particular, arecoline appears to be combined with a pronounced nicotimc action is seen at very high concentrations, e.g. 4 x 10W4M, where an initial C-peak precedes an almost compiete C-paralysis followed by new C-peaks or a progressive C-activation (Fig. 4d). Like the effect of nicotine, the increased activity is quickly reversible in sea water, cf. the slow reversibility of the pure muscarinic effects mentioned
Muscular
activity
in the sea urchin.
I.
3
Table IA and B Effects of nicotituc and muscarinic agents and agents wtth a mtxed effect mcludmg chohnesterase inhibitors on the C-activity and the state of the ammal ctlia. Act : x symbols refer to the degree of stimulation, nntial respective delayed and of longer duration: 0 mdtcates no or very weak stimulation. Tet: x symbols refer to initial tetanic C-activity; 0 mdicates lack of tetamc activity although a marked initial C-peak may occur. Paralys.: x-symbols refer to the occurrence of complete respectively incomplete C-paralysts; 0 indtcates lack of C-paralysis. Late act.: x-symbols refer to the late C-activity, e.g. after C-reactivation, O-symbols refer to prolonged C-paralyses. Cil. paralys : x-symbols refer to prolonged ciliary paralysis; O-symbols Indicate lack of ctliary paralyses although the ciha may show some stgns of tmtation; x to 0 Indicates temporary ciliary paralysis until C-reactivation occurs Agents
Act.
(a) Ntco. IO-P-10-s 10-6
xx x to xx
TMA 10 -’ 10-J 10-e
Paralys.
Tetany
:
Late act. 0 xx
:
xx
X
t :;
c) 0
;r 0 0 0
Cil. paralys.
;,
0 xx 0
X
X
t”x,
0 0 0
x to 0 0
DMPP 10-s 10-4 10-b
;x;
0 0 0
PTMA 10-1 10-4 10-6
t:; 0
0 0 0
0 0 0
;x, 0
0 0 0
0
0
0
0
0
xx
0
(0)
xx
0
xx
tb) Mow.* 4 X 10 4-10-6 Polo and areco 4 x 10 ’
‘Must. refers to muscarme, oxotremorme, bethanechol, metachohne and also to ptlocarpme and arecohne from 10~4 M downwards Agents (a) Carba. 10-s 4 X 10-q 10 -4 10-b ACh 10-s 4 X 10-4 10 s 10-b (b) Physo. 10~~4 10-s 10-e 10-1 10--s
Act. xx xx
Tetany.
Paralys.
X
X
0
x
X
X
;,
;,
: 0 0
tt”x,, 0 0
XX XX
xx xx : xx xx xx
Late act.
xx
X
x x
x
X
Brief
0 xx
Late Dechne Dechne High P Decline Dechne
: xx x Late xxx Tetamc xxx Tetamc 0
Cil paralys x x (to 0) x to 0 0
K$ 0
x to 0 x (to 0) 0 0 0
0
:,
;I 0 0
BW 284 C 51 ]0~~4-]0-6 10-7
0 x too
0 x too
0 0
0 0
0 0
Mipafox 10-4-10~s 10-6 10-s
0 (x1 x to 0
0 0 0
0 0 0
0 t(x)) 0
0 0 0
XX
in a later section. Arecoline also affects the cilia, although complete paralysis does not occur. Agents with combined strong nicotinic and muscarinic activity Carbachol has a nicotine-like effect. There are, however, some important reservations to this, cf. Table lB, a (also Fig. 9 in Gustafson and Treufeldt, 1981). The C-paralysis which follows the initial tetanic burst may indeed be permanent or of very long duration, but only at concentrations down to 4 x 10e4 M, and C-paralysis for more than 15 min only occurs down to lo-’ M. Furthermore, contrary to the case with C-paralyzing nicotine solutions, the
to 0
C-paralysis is attended by very pronounced S- and L-activity. In addition, once the C-movements reappear, they are in general extremely frequent and of high amplitude and foreshadowed by the closely related P-movements. C-paralysis is attended by ciliary paralysis, but the cilia are reactivated in connection with the C-reactivation. The muscular reactions suggest that the strong nicotine-like action of carbachol is moderated by its muscarinic effect. This is in line with the response to carbachol 10e6 (cf. Fig. 9 in Gustafson and Treufeldt, 1981). In this case the strong C-activation declines but without a parallel decline in the amplitude of the C-movements, i.e. contrary to the low salinity
TRYGGVECUSTAFSON
Fig. 2. Effects of mcotinic agents at normal salinity. (a) Nicotine IO-’ M (b) Nicotine 10m6M (c) TMA 10~’ M (d) DMPP 10 -4 M Y-axls. Frequency of movements; X-axls: Time m hrs; for further definitions see section “Diagrams”. C-activity indicated by round dots, S-activity by f signs, L-activity by rhombic dots, C-curve also marked by a C. T indicates tetanic C-activity. response to nicotine 10 -’ in Fig. 3, I.e. the decline apparently not due to overstimulation but to muscarimc calming down. The muscarinic action carbachol may also be responsible for the lack prolonged C-paralysis at very high concentrations
IS a of of of
Fig. 3. Effects of nicotine 10e4 M at a reduced salinity, 21 promille. Y- and X-axis and symbols for C-. S-, and L-activity defined m legend to Fig. 2.
carbachol after premcubatlon of the larvae with low concentration of the same agent (cf. Figs 4 and 8 in Gustafson and Treufeldt, 1981). Such an “adaptive” effect has so far never been observed in similar experiments with nicotine. The spontaneous C-reactivation after carbachol paralysis may also reflect a muscarine-induced “adaptation” (cf. Fig. 9 in Gustafson and Treufeldt, 1981 and the effect of strong pilocarpine and arecoline). Stimulation and C-paralysis is quickly reversible in sea water and repeated exposure to carbachol brings about renewed paralysis provided the previous exposure was not too long (cf. Fig. 7 in Gustafson and Treufeldt, 1981). As in the experiments with nicotine, the dose-response relations depend on the salimty. At 21 promille, the effect of carbachol4 x 10s4 M is similar to that of carbachol 10m6M at high salinity whereas carbachol 10e6 M is only slightly stimulatory. The reactions are the same in very young larvae. The C-stimulation is always strongest in well pigmented larvae. In experiments with acetylcholme, fresh solutions were frequently applied during the course of the recording. The effects are summarized in Table 1B, a. One may suggest that the nicotinic effect of acetylcholine is strongly moderated by its muscarinic action as long C-paralysis only occurs at a very high concentration, 10e3 M. In addition, this paralysis is attended by high P-activity, a reaction closely related to the C-movements (Fig. 5a). At 4 x 10m4 M there is at most an extremely brief C-paralysis. The C-activity rises to very high values after which it declines to a
Muscular
activity
in the sea urchin. 1. (d)
Fig. 4. Effects of muscarinic agents. (a) Muscarine Philocarpine 10e4 M (d) Arecohne 5 x 10m4 M. Transfer symbols for C-. S-, and L-activity
moderate level, presumably due to a progressive muscarinic action since the amplitude of the C-movements is unchanged (Fig. Sb). At lo-’ M and lower concentrations there is at most a very brief activation followed by a muscarine-like calming down (Fig. 5~). The vigorous stimulatory effect of acetylchohne 1O-4 M is strongly counteracted if the larvae are pretreated with low concentrations of this agent. One may assume also that this “adaptation” reflects the muscarinic effect during pretreatment. Ciliar paralysis only occurs in connection with C-paralysis. The C-stimulation is reduced at very low salinity but is favoured by high pigmentation. The effects of proprionylcholine and buturylchohne are similar to that of acetylcholine. Cholinesterase
inhibitors
The effects of three cholinesterase inhibitors, physostigmine, mipafox, and BW 284 C 51 (benzyldimethylguanidine, Wellcome Research Laboratories) have also been investigated. The action of physostigmine was described in an earlier report (cf. Fig. 3 in Treufeldt and Gustafson, 1981), but after this some further data have been collected (cf. Table 1B, b). At 10e9 M the effect is a clear muscarinic one, as at low concentrations of acetylchohne, but already at concentrations around IO-’ M there is a vigorous, nicotine-like C-activation, gradually characterized by intermittent tetanic periods at the brink of paralysis but never going beyond it, presumably saved from this by the indirect muscarinic activity. At lo-‘M, however, the effects are similar to those of strong nicotine and carbachol. The results thus suggest that physostigmine, apart from its action as a
2 x 10e4 M (b) Oxotremorine 5 x 10e5 M (c) to pure sea water at arrow B. Y- and X-axls and defined in legend to Fig. 2.
chohnesterase inhibitor, has a direct nicotine-like effect on a superficial receptor (cf. a later section). That the stimulation as well as the paralysis is quickly reversible upon early interruption of the treatment is in line with this assumption. The C-paralysis by physostigmine is followed by strong reactivation and reappearance of ciliar activity. This change is considerably quicker at 1O-4 than at lo-’ M which suggests that the muscarinic effect of the accumulation of acetylcholine moderates the nicotine-like action. That the action of physostigmine after 1 hr of treatment at 10m4 M is only slowly reversible in sea water no doubt reflects a sustained acetylcholine effect (cf. Fig. 21 in Gustafson et al.. 1972b). At low salinity, the paralysis induced by physostigmine 10e5 M IS replaced by a vigorous peak in C-activity as in similar experiments with strong carbachol. The effects of the two other cholinesterase mhibitors, BW 284 C 51 and mipafox, differ markedly from that of physostigmine (cf. Table IB, b). This suggests that they are entirely due to the accumulation of acetylcholine, the nicotine-like effect of which is moderated by its muscarinic activity. A rather brief, initial stimulation has been recorded after exposure to BW 284 C 51 lo-‘M, at least at the onset with some tetanic tendency, particularly pronounced at high salinity (Fig. 6a). At higher concentrations, the C-activity is comparatively low, although a brief initial peak has once been recorded (Frg. 6b). Unquiet larvae are calmed down. In spite of the low stimulatory activity that dominates the picture, it is evident that the larvae are steadily affected as the C-movements occur in swarms or even slight bursts separated by calm periods. The ciliary state is unaffected, even
TRYGGVE GIJSTAFSON (a)
b)
Fig. 5. Effects of acetylcholine. (a) lo-’ M; note the P-activity, curve marked P. (b) 4 x 1O-4 M (c) 10e5 M 2 hr Y- and X-axis and symbols for C-. S-, and L-activity defined in legend to Fig. 2.
at the peaks in activity. The effects of mipafox within the concentration range 5 x 1O-4-lO-8 M reminds of those of BW 284 C 51, although no tetanic tendency has been observed.
Fig. 6. Effects of BW 284 C 51. (a) IO-’ M (b) 10m5 M Y- and X-axis and symbols for C-, S-, and L-activity defined in legend to Fig. 2. T indicates tetanic C-activity.
Nicotinic antagonists In order to shed some further light on the relationship between the nicotinic action of an agent and its C-stimulating respective C-paralysing ability. the effects of agents that block or inactivate the nicotinic receptor were studied, i.e. those of conventional nicotinic antagonists (Table 2), two snake toxins, and DTT (dithiothreitol), an agent that reversibly inactivates the nicotinic receptor (cf. Karlin et al., 1973). Most of the experiments were restricted to the concentrations used to counteract the nicotinic effects (see a later section). The results of the experiments with a long series of quaternary nicotinic antagonists at high concentrations, 10-4-10-3 M, are very consistent and may be illustrated by Figs 14 and 15 in Gustafson et al. (1972b) referring to succinylcholine 5 x 10e4 and decamethonium 10e4 M and to the lower curve in Fig. 7 in the present paper. An initial, brief and weak stimulation may occur here, followed by low activity. Spontaneously unquiet larvae are calmed down and the cilia remain active. At very low concentration, around 10m8 and lo-‘M, the stimulatory
Muscular Table
2. Effects
physostlgmme
of antmlcotmlc as revealed
actlvlty agents
in
(Ant!
by the quickness
the
sea
urchin.
1
) on the response of C-reactwatlon
to mcotine,
carbachol.
and the followmg
and
mtenslty
of
the C-movements 10m4
Nm Agents
Carba
+Antl
Decamethomum
2x
Succmylchohne
4 x 10~4
Carba
+Antl.
10~4
xx
IO
4 xxx
10-J
x
IO
J XIX
5x10 10-4
5x10 5x10
Gallamme
IO
5x10
4 xxx 4 xx
a xx
Arfonad” 10-J
(X)
x
5 x 10-J
xx
5 x 10-4
xx
5x10 IO
“Trlmetaphan
x. xx. and TXX mdvzate cle.u, column
IS based
C-paralysis counteractmn +
mdlcates
sumlar
strong
admrxed
and very strong wth
effect Induced
effect
to those of the other
of varymg
antnncotmlc
effect of decamethonium is considerable, however. but quickly fades upon increase in concentration. A similar nicotine-like stimulation has been observed with low concentrations of succinylcholine. another depolarizing antimcotme. The effects of the tertiary antinicotines pempidine. mecamylamine, and benactyzine at 10-5-10-“M are similar to those of the quaternary agents. The effect of tubocurarine is a clear exception to the general rule. At 10e4 M there is generally an enormous activation, gradually dominated by Cmovements (Fig. 7, upper curve). In fact. it IS similar to that of serotonin (cf. Gustafson et al., 1972a). It is quickly reversible in sea water. A similar stimulatory effect but of shorter duration has been recorded at high concentrations of arfonad and, in a single case, also with decamethomum. When a strong C-activation occurs, It 1s always attended by the appearance of granules in the esophagus that swell and collect in the stomach. When such granules do not appear, the effects of the agents concerned follow the normal antinicotinic pattern. a-Bungarotoxin lo-‘M has a certain stimulatory effect. in particular to the S-activity. At IO-’ M a brief initial C-peak has been seen. The effect of cc-najatoxin within the same concentration range is
ok,,,,,,,,,,,,,,,,,,,,,,, I
2
3
d
I+
10-d
I+
5 x 10-4
I+
IO_’
xxx
1+
2 x 10~4
I+
4 xx
5 x IO_’
1-t
4 XT
5x
10
5x
10-h
antlparalyuc
effect
strategy
(antrmcotme
a varymg
to the physostlgmme
of the stlmulatory an antlpardlytlc
I+
5 x IO_’
5 1-t I+
camsylate
on expenments
or dwxtly
+ AntI.
5 x 10-J
xx
5 x 10-J
Pempdme Mecamylamme “Tetraethylammonlum.
xxx 4 xxx
10-4
Alloferm Myrolon
Phvso
(xx IO_’
Hexamethonlum TEA” Chlorrsondamme
-
do. + Ant!.
5
Fig. 7. Effects of decamethonium and tubocurarine. Upper curve: C-activity induced by tubocurarme 10m4M. Lower curve: C-activity Induced by decamethonium 10m4 M. Y- and X-axls defined m legend to Fig. 2.
solutmn)
I (Inhlbltmn)
by low concentrations mtenslty
The physostlgmme
The
admlxed
after
symbolizes
of physostlgmme.
effects of tubocurarme
1s
agents
similar as is DTT 10-j and 2 x 10m3 M. The reaction between the agents concerned and the nicotinic receptor thus appears to bring about a certain, more or less transient. activation. The state of the cilia at the animal plate. however, IS unaffected. Attenuation agents
qf the nicotinic
effects
by antinicotinic
As most of the antinicotinic agents treated in the preceding section do not have any pronounced or at least no persistent stimulatory effects at appropriate concentrations, they could be used as tools in a further analysis of the nicotinic effects. The experiments were carried out with varying strategy (cf. Table 2). As certain antinicotinic agents may have side effects, even muscarinic ones, it was important to use a wide variety of these compounds. However, their effects on the response to nicotine-active agents are quite consistent (cf. the Table). Upon simultaneous exposure to nicotine 10m4 M and nicotine antagonists, the C- and ciliary paralysis is counteracted, instantaneously or with short delay (cf. Fig. Sa, b). When the C-reactivation is delayed. it is preceded by marked S- or L-activity or both. The C-reactivation also occurs if the antagonists are added to larvae already paralyzed by pure nicotine. The effect is weaker, however, which suggests that the paralytic effect of nicotine is cumulative. In experiments with carbachol 4 x 10m4M, the results are analogous, although the C-reactivation is often preceded by marked P-activity. After pretreatment with antagonists before the admixture of carbachol, the paralysis fails completely. In experiments with decamethonium, a concentration of lo-’ M is enough to reactivate the C-movements of larvae in the presence of carbachol lo-’ M, instantaneously and very strongly. In fact, the most powerful effects are always seen when the concentration of the antagonist is below that of carbachol (cf. Fig. 9). A prerequisite for high C-activity is evidently that the remaining nicotinic effect is rather high. The strong decline in C-activity, without decrease in the amplitude of the C-movements, that
TRYGGVE GUSTAF~ON
Fig. 8. Effects of antimcotinic agents on the response to mcotine and carbachol in C-paralyzmg concentrations. (a) Decamethonium 10e4 M + nicotine lo-’ M (b) Succinylchohne 10mJ M + nicotme 10m4 M (c) Effects of pure carbachol 4 x lo-” M, added at the arrow after premcubation with pure succinylcholine lo-) M for 1.25 hr showing quick reversibility of the antinrcotinic effect; some P-activity IS shown in the curve marked P. Y- and X-axis and symbols for C-, S-. and L-activity defined m legend to Fig. 2. Curve indicated by triangles and P refers to P-activity. T indicates tetanic C-actlrity
occurs after a period of reactivation, may be due to the muscarinic effect of carbachol. The effects of the antinicotmes are quickly reversible. The reversibility may lead to complete paralysis m pure carbachol (Fig. 8~). However, in larvae which have been subjected to prolonged treatment with a mixture of carbachol and an antinicotine, paralysis fails upon exposure to pure carbachol, supposedly due to the muscarinic effects of carbachol during the preceding exposure (cf. an earlier section). The muscarinic effects will be further elucidated in a later section. a-Bungarotoxin 6 x lo-‘M counteracts the Cparalysis of admixed carbachol 4 x IO-“ M (Fig. 10a). The toxin effect is also distinct at lo-‘M, although the C-reactivation is replaced by the P-movements forerunners of C-activity, i.e. (Fig. lob). The cihary paralysis is also counteracted to a varying extent. A clear antiparalytic effect re-
mains after transfer to pure carbachol. The antiparalytic effect of a-najatoxin is presumably weaker than that of a-bungarotoxin, at least it is more quickly reversible after transfer to pure carbachol, although the antiparalytic effect on the cilia persists. DTT 10m3 M also prevents the C-paralysis and to some extent also cihary paralysis. The protective effect is almost completely reversible in pure carbachol. That physostigmine, apart from its action as a cholinesterase inhibitor, appears to have a direct nicotine-like effect is supported by numerous experiments (cf. Table 2). A long series of antinicotines thus counteract the stimulatory effect of low concentrations of physostigmine, its paralytic effects at lo-’ M (cf. Fig. II), and also the high activity of larvae spontaneously C-reactivated in physosttgmine 1O-4 M. The C-paralyzing effect of nicotine, carbachol, and physostigmine is not counteracted if the agents act
Muscular activity in the sea urchin. I.
Fig. 10. Effects of ~-bungarotoxin on the response to a paralyzing concentration of carbachol; P-activity is shown
Frg. 9. Effects of decamethonium on the response to a normally C-paralyzing concentration of carbachol; diagram restricted to the C-activities; curve 1: De~methonium 2 x 10e4 M + carbachol 4 x 10e4 M, curve 2: Decamethonium 4 x lo-“M +carbachol 4 x 10e4M. Y- and X-axis defined in legend to Fig. 2.
together at high concentrations, but lobeline and PTMA decrease the paralytic effect of nicotine. It thus appears that less effective nicotine agonists have a certain capability to displace nicotine from its receptor. Attenuation of the nicotinic effect by muscarinic agents In some previous sections, it was suggested that the nicotinic effects of agents such as carbachol, acetylcholine, and physostigmine are moderated by the, direct or indirect, muscarinic effects. One may therefore expect that the paralytic effects of nicotine would also be weakened or turned into strong stimulation in the presence of pure muscarinic agents, acetylcholine, BW 284 C 51, mipafox, respectively very low concentrations of physostigmine, and that such agents would further reduce the paralytic effect of carbachol and physostigmine. The results of a great number of experiments are in line with this expectation (cf. Table 3 and the examples in Fig. 12ax). They also show another prominent regularity that is quite illuminating (cf. the following). The maxima1 C-activity upon exposure to a mixture of nicotine and agents with a pure or dominating muscarinic activity depends on their proportions (see Table 3 and Fig. 12b and curve 2 in d). It is much higher at rather low concentrations of the muscarineactive agents concerned than at higher concentrations. It is therefore clear that high muscular activity is favoured by a proper balance between the nicotinic and the muscarinic signals. A similar conclusion can
curves P. (a) cc-bungarotoxin in the marked 6 x lo-’ M +carbachol 4 x 10e4 M (b) a-bungarotoxin IO-’ M + carbachol 4 x 10S4M after 5.5 hr pretreatment with pure toxin solution. Y- and X-axis and symbols for C-. S-, and L-activity defined in legend to Fig. 2.
be drawn from the experiments with nicotine or carbachol plus cholinesterase inhibitors (cf. curve 1 in Fig. 12d), but the nicotinic contribution of the acetylcholine piled up should not be disregarded. The role of the concentration relationships is also illustrated in Fig. 12b where the temporal changes in the C-activity is plotted in two nicotin~oxotremorine experiments. It is evident that the C-activity drops when it has reached a maximum (cf. also the decline in Fig. 12a), and that the decline is accentuated when the oxotremorine concentration is increased from lOas to low4 M as in Fig. 12b. One may compare this with the effects in Fig. 9 which in fact also illustrate the activities at varying nicotinic and muscarinic provocation. However, in experiments with stronger nicotine concentration, 2 x 10M4M, a high C-activity is reached also at very high concentration of oxotremorine, 4 x 10e4 M, but here also there is a sharp decline (Fig. 12~). The antiparalytic effect of muscarinic agents and the characteristic time-course of the C-activity is also
Fig. 11. Effect of alloferin 2 x 10m4M on the effects of physostigmine at stimulatory and paralyzing concentrations. Pretreatment with pure alloferm for 45min; physostigmine 5 x IO-‘M admixed at first arrow; physostigmine 10m5M admixed at second arrow. Y- and X-axis and symbols for C-, S-, and L-activity defined in legend to Fig. 2.
-9
-8
-7
-6
-5
-4
Fig. 12. Effects of muscarmic agents mcluding mipafox on the response to nicotine-acttve agents at C-paralyzing concentrations. (a) Oxotremorine 4 x IO-” M -I-carbachoi 4 x tom4 M (b) 1. C-activity at oxotremorine lo-’ M c nicotme 10e4 M; oxotremorine concentration increased to 10m4M at the thick arrow; 2. C-acttvity at oxotremorine low4 M + mcotine IO-” M. T-symbol for tetanic C-activtty only refers to the upper curve (c) Oxotremorine 4 x lO-4 M fnicotine 2 x lo-” M (d) The maxtmal C-activity reached upon exposure to C-paralytic nicotinic agents plus muscarinic agents in molar concentrations indrcated by exponents at the abscissa. Curve 1. Mipafox fcarbachol 4 x 10e4 M; curve 2. Oxotremorme + nicotine 10e4 M (e) Slow reversal of the betanechol effect after exposure to betanechol 6 x 10m4M for 2.5 hr; pure carbachol 4 x 10-4M added at the arrow. Y- and A’-axis and symbols for C-, S-, and L-actrvtty (in diagrams a-c and e) defined in legend to Fig. 2 T Indicates tetamc C-activity.
Muscular activity In the sea urchin. I. evident in experiments where physostigmine 10e5 M is combined with strong betanechol. The powerful stimulation brought about by low, non-paralyzing concentrations of physostigmine also decreases upon admixture of betanechol and also the spontaneous C-reactivation level in physostigmine 10m4M. The muscarinic effects in these experiments instantaneously reversible (cf. Fig. 12e).
are not
Antlmuscarines
As the muscarmes in the results reported counteract the effects of mcotinic agents, it may be supposed that a nicotine-muscarine interaction also plays a role in the intact larva, and that the muscarinic function serves as a “brake” that counteracts excessive activity. If so, one could expect that antimus-
carinic agents may release a high muscular activity. The effects of a series of antimuscarines at high concentrations are in line with this assumption. Atropirie at concentrations down to at least 5 x 10e5 M thus bring about a very strong C-activation, often with some delay. A clear stimulatory effect has also been recorded with scopolamine 5 x 1O-6 M. At low salinity the effect consists of a long series of peaks which, in the long run, tend to decrease in height as well as in their duration (Fig. 13a). At high salinity the peaks fuse into a block followed by a strong decline in activity or even total paralysis (Fig. 13b). The effects of atropine and scopolamine are reproduced by methylatropine and oxiphencyclimine. The reversibility of the antimuscarine effect is quick, except when total paralysis has been reached.
3%.
300.
11
(b)
2s
zoo 150
loo
50
0
(d) ic
Fig. 13. Effects of atropine and its counteraction by a muscarinic agent. (a) Atropme lOa M at a sahnity of 21 promille (b) Atropine 10m4M at normal salinity (c) Atropine lOA M + betanechol 10e4 M after pretreatment with betanechol 10m4M for 2.5 hr; atropine admixed at the arrow. Y- and X-axis and symbols for C-, S-, and L-activity defined in legend to Fig. 2. T indicates tetanic C-activity.
TRYGGVEGUSTAFWN
12
(b)
iic
.
I i
I l
. Pi
l \
\.
Fig. 14. Effects of atropme on the response to mcotine and physostigmme. (a) Atropine 5 x 10m4 M + nicotine 10m4 M (b) Atropine 10m4 M + physostigmine 10m5 M; note the scale. Y- and X-axis and symbols for C-. S-. and L-activity defined in legend to Fig. 2. T indicates tetanic C-actwity
This indicated that the agents interfere at a superficial level. That their effect is counteracted by oxotremorine. betanechol, and by low concentrations of BW 284 C 51 and physostigmine may suggest that muscarinic receptors are involved (cf. Fig. 13~). That the antimuscarines may have other effects as well should not be neglected, however (see the following). If the antimuscarines only weaken the muscarinic “brake”. the admixture of nicotine low4 M would easily lead to overstimulation followed by C-paralysis. This is partly in line with the results of the experiments carried out (cf. Fig. 14a). The further course of the activity curve, however, suggests that atropine at high concentrations has a certain antinicotinic effect as some C-reactivation occurs. With isopropamide. another antimuscarine. the antinicotinic effect is considerably stronger. The paralytic effect of carbachol 4 x 10e4 M is also effectively counteracted by the whole series of antimuscarines. An unprecendently intense stimulation of long duration is triggered in experiments with atropine 10m4 M plus physostigmine 10e5 M (cf. Fig. 14b, note the scale). One may think that the paralyzing nicotine-like effect is here decreased into a preparalytic and therefore strongly stimulatory intensity due to the antinicotinic effect of atropine which, at the same time, retards the muscarinic calming down. DISCUSSION
The pattern of contractile activities in the intact sea urchin pluteus is rather regular and monotonous (cf. Fig. 4 in Treufeldt and Gustafson, 1981). One may therefore assume that a pacemaker-function is involved. Indeed, there may be two different pacemakers, one with a comparatively high basal frequency and only responsible for the muscular
activity, one with a low frequency (around 8 impulses per hr) controlling the muscular as well as the intestinal movements-the stomach and proctodaum cycles (cf. Fig. 1 in the present paper and Figs 6. 8. and 9 in Gustafson et al., 1972a). Both pacemakers may be further activated and paralyzed in a similar way, although their relative role may vary in the course of an experiment (cf. Treufeldt and Gustafson. 1981). An interpretation of the organization of the pacemaker-system is presented in Fig. 5 in the report just quoted. The results so far reported indicate that the nicotine-active agents are stimulatory and that Cparalysis as well as ciliary paralysis, that immediately follows a vigorous, tetanic, peak in C-activity, IS the result of excessive stimulation. Agents wtth pure muscarinic activity, on the other hand, keep the muscular activity low, calm down spontaneously unquiet larvae, and do not affect the ciliary state. The rapid effects of the agents concerned and their antagonists indicate that they interfere with superficial receptors involved in the muscular as well as in the ciliary control. The quick reversibility of the nicotmic agents is in line with this. The delayed reversibility of the muscarinic effects will be discussed later. As the dose-response relation of purely nicotinic agents as well as that of carbachol is changed at lowered salinity, one may assume that the nicotinic effects are mediated by an increased influx of some ions, probably Ca’+ (see a later report in this series). The muscular and ciliar paralysis may therefore reflect an excessive ionic influx. As the muscarinic effects on unquiet larvae may be mimicked by reducing the salinity, it is logical to conclude that the muscarines decrease this influx (see a forthcoming report). Due to this action they may counteract nicotinic stimulation and paralysis.
Muscular activity in the sea urchin. I. That the effect of carbachol and, in particular, that of acetylcholine differs from that of nicotine is in line with the fact that these agents, in many systems, have nicotinic as well as muscarinic effects. In the sea urchin pluteus, the muscarinic effect of acetylcholine is apparently more strongly expressed than that of carbachol. However, the muscarinic effect of carbachol is clearly manifested when antinicotines are admixed (Fig. 9), cf. also the decline in C-activity without decrease in C-amplitude at low salinity. It has already been suggested that the “adaptive” effect during exposure to carbachol, including the spontaneous C-reactivation, reflects a muscarinic action. A general impression is that the threshold for the muscarinic effect in the pluteus larva is much lower than that of the nicotmic agents. In line with this, the effects of low concentrations of acetylcholine are predominantly muscarinic. When agents with pure nicotinic and muscarinic activity are combined, the ratio between the two effects is of decisive importance, both for the maximal C-activity reached after paralysis and for the further course of the activity curve. The role of this ratio is also illustrated in experiments with carbachol-treated larvae exposed to varying concentrations of antinicotines (Fig. 9). The effects of the cholinesterase inhibitors BW 284 C 51 and mipafox are evidently mediated by acetylcholine accumulation. Its nicotinic stimulation is here hampered by its muscarinic effects. The action of physostigmine, on the other hand, suggests that this agent also has marked, direct nicotinic effects. This is in line with the effects of antinicotines on physostigmine-treated larvae. In the long run, which is particularly clear at high concentrations, its nicotinic effect is moderated by the acetylcholine accumulated. The antiparalytic effect of all the cholinesterase inhibitors at low concentrations are evidently due to the indirect muscarinic effect. The results m Fig. 12e and a series of similar experiments indicate that the muscarinic effects, e.g. on the guanylate cyclase system, are not immediately reversible. One may ask if this reflects an after-effect on this system. It may be added that the muscarinic effect of oxotremorme is strongest at high salinity, an observation that might reflect the importance of Ca’+-ions in the activation of the cyclase. The C-activity in a larva paralyzed by TMA sooner or later reappears, in spite of the lack of direct muscarinic activity (Fig. 2~). This could depend on acetylcholine released from an inner source (see the end of the discussion). However, if the excitation is mediated by an influx of Ca’+-ions, this may also lead to an activation of the guanylate cyclase system, a presumed target for the muscarinic action. Such a mechanism. however, is apparently insufficient to reactivate larva paralyzed by very strong nicotinic treatment. The role of the guanylate cyclase system will be further discussed in a forthcoming paper in this series. That the activity of an intact larva is governed by a certain muscarinic “tonus” is suggested by the strong stimulatory effects of antimuscarines at high concentrations, an effect which is counteracted by muscarines. One may say that this “tonus” serves as a “brake”. However, the antimuscarinic action appears to be contaminated by an antinicotinic effect. It
13
may even reflect direct effects on some ionic channels. The different muscular elements are all derived from the celomic rudiment (cf. Fig. 1 in this paper and Gustafson and Wolpert. 1963). One may therefore ask how one type of muscular activity may dominate at the expense of the others, and why it even may be monopolized. A preliminary working hypothesis is that the different muscular elements have different thresholds of excitation. The C-movements have a relative long duration and are also more forceful than the twitch-like L-movements, sometimes even tetanic. They may therefore require very powerful signals. One may also suggest that these signals somehow suppress the signals to the other muscular activities, cf. the S-favouring effect of the muscarines that decrease the C-activity and the opposite effects of serotonin and some catecholamines described in Gustafson er al. (1972a) and later reports in this series. In addition, a very high L-activity may make the peristaltic system incapable of producing the concerted, consecutive L-twitches involved in the peristaltic waves denoted by S-movements, cf. heartflimmer. The C-stimulatory effect of nicotinic agents is particularly strong in pigment-rich larvae. The relationship between the pigment formation and the monoamine metabolism demonstrated by Asashima (1972a, b) may therefore indicate that the C-stimulation due to an increased ionic influx, e.g. induced by nicotine, is mediated or amplified by an inner monoaminergic system. The main anatomical localization of this system is, hypothetically, the mouth region (cf. Fig. 5 in Treufeldt and Gustafson, 1981). a site that, according to histochemical investigations, does contain monoamines (cf. Fig. 5 in Ryberg, 1974). Monoamines released from this system may also be responsible for the suppression of the S-activity, cf. the effects of serotonin and various catecholamines (Gustafson et uI., 1972a) and later papers in this series. That the release may be governed by acetylcholine is in line with the observation that the monoamine containing structure is closely related to, or possibly identical with, high specific cholinesterase activity (cf. Fig. 26c in Gustafson et al., 1972b). The aberrant monoamine-like effect of tubocurarine etc., which is correlated with the release of some granules into the esophagus, may be related to a massive release of monoamines from the site just discussed. In any case there are metachromatically staining cells in its vicinity that may be the source of the granular material that appears in connection with strong C-stimulation by these agents (cf. Ryberg and Lundgren, 1975). That tubocurarine and some other antinicotinic agents at high concentration may release pharmacologically very active agents is well known indeed (cf. Goodman Gilman et al., 1975). The signal system controlling muscular activity in the pluteus is apparently closely related to the one controlling ciliary activity in the embryo (cf. Soliman, 1983a, b, 1984a, b). An important question is how the embryonic system develops pacemakers with a comparatively slow rhythm. Closely connected with this question is how strong a nicotinic stimulation may turn off the pacemaker-signals eliciting C-activity. One may speculate that the calm periods between the
14
TRYGGVE GUSTAFSON
pacemaker-bursts and the long paralysis after strong nicotinic stimulation reflect the same mechanism, e.g. a hyperpolarizing outflux of K+-ions induced by the influx of Ca2+ (cf. Meech, 1980). This question will be further discussed in later reports in this series. In conclusion, the muscular and cihary activity in the sea urchin pluteus is affected by nicotinic as well as muscarinic agents. The nicotinic effects are stimulatory, but excessive stimulation is quickly followed by paralysis, in particular in the most powerful muscular strands. Muscarinic agents, on the other hand, have a calming effect and counteract the action of nicotinic agents. Acetylcholine, which has nicotinic as well as muscarinic effects and which occurs in the larva. is presumably involved in the control of activity of the normal larva. The cholinergic system within the larva can evidently be affected via a receptor system at the larval surface. It is suggested that the cholinergic signals bring about a release of monoamines that amplify the signals to the powerful C-activity and at the same time suppress the other muscular activities. It is also suggested that the paralytic effect of strong mcotinic treatment refect a hyperpolarizing outflux of K+ induced by a strong influx of Ca’+ which may play a central role as a charge-carrying ion. Acknorcledgements-This work has been supported by the Swedish Natural Science Foundation. I am indebted to the head of the Kristineberg Marine Biological Statton, Professor J.-O. Stromberg, and to hts staff for their never fatling generosity. I also express many thanks to Rein Treufeldt for hts patient assistance in a great number of expertments and also to docent Jan Nedergaard for his constructtve crttictsm during the preparation of the manuscript.
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