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Pharmacological control of ciliary activity in the young sea urchin larva: Chemical studies on the role of cyclic nucleotides
Cony.
Biochem.
Printed in Great
Phvsiol.
Vol. 78C, No. I, pp. 175-181, 1984 c
B&in
0306-4492/84 $3.00 + 0.00 1984 Pergamon Press Ltd
PHARMACOLOGICAL CONTROL OF CILIARY ACTIVITY IN THE YOUNG SEA URCHIN LARVA: CHEMICAL STUDIES ON THE ROLE OF CYCLIC NUCLEOTIDES SHERIF S~LIMAN The Wenner-Gren
Institute
for Experimental Biology, University 45 Stockholm, Sweden. Telephone:
of Stockholm, 08-34-0860
Norrtullsgatan
16, S-l 13
(Received 29 June 1983) Abstract-l. Distinct peaks in CAMP and cGMP content during early development, partly opposite to each other, may be correlated with the two main phases of gastrulation and ciliary activity. 2. Monoamines increases CAMP formation. A transient or extended decrease follows, presumably reflecting some feedback mechanism. Muscarinic agents and CaZ+ interfere. 3. The developmental variation in cyclic nucleotides may reflect a temporal shift in the role of various signal substances as well as feedback regulation related to Ca2+ influx. 4. The opposite changes in CAMP and cGMP during early gastrulation may reflect a mutual dependency of the two nucleotide cyclases related to changes in Ca2+ influx.
INTRODUCTION
morphogenetic cell activities are both closely related to the cellular level of ionic calcium in some important compartments. The results can also be considered as a supplement to the studies on the neuropharmacological control of muscular activity in the pluteus larva carried out by Gustafson and Treufeldt (1981) and Treufeldt and Gustafson (1981) and may hence contribute to the elucidation of the problem of ontogeny of behaviour as ciliary activity starts before muscular activity, although the control mechanisms may be partly the same.
In earlier papers in this series, the control of larval ciliary activity was approached by studies on the ciliary effects of cholinergic and monoaminergic agonists and antagonists (Soliman, 1983a,b). As a further step in the analysis, the effects of cyclic nucleotides and calcium ions in connection with ciliary activity was investigated since they may be presumed to mediate the agonist effects concerned (Soliman, 1984). In this paper the results of chemical analyses of cyclical nucleotides are documented, both with regard to developmental changes in normal larvae and to the effects of various agonists and alterations of the calcium content of the sea water. Although some of the results of the present study may still be regarded as preliminary, in particular with respect to some changes in cGMP, the observations support the idea that calcium is involved in ciliary regulation and that the cyclic nucleotides have a central role in the membrane flux of calcium ions and vice versa, i.e. that cyclic nucleotides and calcium ions have a mutual relationship as in many other systems more extensively studied (cf. Rasmussen, 1981). The results also contribute to the discussion on the so-called phase specificity of different physiological signal substances in early development (cf. Gustafson and Toneby, 1970, 1971 and Gustafson and Treufeldt, 1981), and may shed some light on the mechanism behind the often drastic fluctuation in the response to agonists as development proceeds, a phenomenon that may overshadow their physiological phase specificity (cf. Soliman, 1983a, 1983b). The final goal of the investigation is to shed some light on the morphogenetic cell activities during gastrulation which attend the start of the ciliary activity and its further changes to a maximum, a minimum and a renewed peak. In fact, it is attractive to think that the ciliary control and the control of
MATERIALS AND METHODS Young sea urchin larvae were used, from hatching, up to an early prism stage, and in some experiments, plutei. Most experiments are carried out with the summer spawning species Psammechinus miliaris, the shallow and the deep water forms living at a salinity of 21 or 26 promille and at 32 promille respectively. Experiments with the winter spawning species Strongylocentrotus droebachiensis and Echinus esculentus, after capture reared at a salinity of 32 promille, are also carried out. After artificial fertilization the material is cultured in beakers under gentle stirring at constant temperatures, around 16” and 8°C respectively. The culture density varies between 500 and 1500 larvae per ml depending on which cyclic nucleotide is to be analyzed, the determination of cGMP requiring a higher larval concentration than that of CAMP. The salinity of the sea water used for the cultures is exactly adjusted to 21, 26 or 32 promille depending on the source of the material. Studies on the effects of altered calcium concentration of the medium are performed by admixture of calcium free and calcium enriched artificial sea water to the normal sea water, but rearing of the larvae, up to the start of the experiments, is always carried out in normal sea water. The effects of various agents on the larval content of cyclical nucleotides are investigated by adding the agent concerned, dissolved in sea water followed by pH adjustment, to the larval suspension. The length of incubation varies between the experiments and is defined in the figure 175
176
SHERIF SOLIMAN
captions. The phosphodiesterase inhibitor 3-isobutyl-lmethylxanthine (IBMX) IO-” M is added to the incubation medium unless otherwise stated. Incubation is quickly terminated by rapid hand centrifugation of the culture sample after which the supematant is replaced by chilled extraction medium (0.3 M perchloric acid, 1 mM EDTA and 10% of acidic methanol 0.1 M HCl in methanol). The larvae are homogenized in a Dounce homogenizer in the extraction medium after which the homogenate is kept for 1 hr during continued chilling in an ice bath. Extraction is terminated by centrifugation at 6000 rpm for 10 min after which the supernatant is transferred into another centrifuge tube where perchloric acid is precipitated by adding 8&85~1 of KOH per ml. After centrifugation at 3000rpm for 10min the supematant is collected in a test tube where pH is adjusted to 5-6 by adding 10 ~1 concentrated acetic acid. A volume of 5&100 pl of the final extract is used for the
determination of the cyclic nucleotides. CAMP is assayed according to the method of Brown et al. (1971) or by using a CAMP assay kit from Radiochemical Centre, Amersham, U.K. cGMP is assayed by aid of the Amersham cGMP radioimmunoassay kit.
RESULTS
Developmentul and cGMP
changes in the larval content of CAMP
A considerable number of experiments on the normal developmental changes in the larval content of cyclic nucleotides are carried out, in particular with respect to CAMP which is studied both in warm and cold water spawning species. The experiments are carried out without IBMX preincubation. The cGMP content turns out to be much lower than that of CAMP (cf. Fig. l), and the number of larvae in each culture sample is adjusted to this.
I
Id’pmol
cGMP/103
pmol cAMP/103
larvae
larvae
I8 20 22 24 24 Time after fertlllzotlon
t
28 (hr)
Fig. 1, Changes in content of CAMP and cGMP during early larval development of Psammechinus miliaris. Salinity 26 promille. Arrow indicates the border between Ga, and Ga,. Note that the CAMP content is lOO-fold higher than that of cGMP. The points correspond to mean + standard error of mean of duplicate determinations in 8-15 cultures. CAMP: solid line: cGMP: dotted line.
The changes in CAMP in the summer spawning species Psammechinus miliaris are quite reproducible. A CAMP peak is seen during Ga, , a minimum occurs at the Ga,-Ga, border, and a renewed increase is seen in Ga, leading to a peak (Fig. 1) or to a plateau. In the advanced gastrula, close to a prism stage, the CAMP content shows a steep rise. When sampling starts somewhat later, the decline after this early peak is clear (Fig. 1). There is evidently a striking parallelism between the temporal variation in the larval content of CAMP here described and the developmental changes in ciliary activity described by Soliman (1983a) in the sense that in both cases a Ga, maximum, a minimum around the Ga,-Ga, border and a new peak or at least a rise can be seen. The only difference is the early “post-hatching peak” in CAMP which is not seen in the assay of ciliary activity; it should be mentioned, however, that in certain experiments with neuropharmaca the initial ciliary activity in the controls is fairly high (cf. Soliman, 1984). It should also be noted that the assays of ciliary activity are interrupted in the advanced gastrula more or less before the final steep increase in CAMP since, at this stage, the larvae change their external shape and their pattern of swimming (see Soliman, 1983a). A general parallelism between the larval content of monoamines and CAMP ciliary activity can be noted (cf. Buznikov et al., 1964 and Soliman, 1983a). This parallelism is only lacking during early Ga, where the monoamine content is low, whereas the CAMP content shows an increase, to a peak or a plateau. This suggest that the formation of CAMP not only reflects the fluctuations in the larval content of monoamines but that variation in CAMP and hence ciliary activity is also governed by some kind of temporary, negative feedback regulation. One should also consider that, as gastrulation proceeds, the role of cholinergic signals may increase (cf. Buznikov et al., 1968) which may also affect the adenylcyclase activity, as discussed below. The Ga, changes in the larval content of cGMP are to a great extent opposite to those in CAMP (Fig. 1). In Ga,, on the other hand, the two cyclic nucleotides show a rather coordinated rise. The Ga, pattern suggests a mutual dependency of the two nucleotide cyclases, e.g. a strong influx of Ca*+ induced by CAMP may hamper the adenylcyclase activity whereas it promotes the guanylcyclase which, via cGMP, hampers the influx of Ca*+ and therefore promotes the adenylcyclase, etc. In Ga, one may presume that the increase in acetylcholine (cf. Buznikov et al., 1968) brings about a muscarinic activation of the guanylcyclase which still may have a positive effect on the adenylcyclase (cf. the preceding paragraph). At the end of Ga,, the inverse relationship may appear again although the experiments are fragmentary. Effects of a phosphodiesterase content of CAMP
inhibitor on the larval
The results presented in the previous section are carried out in the absence of a phosphodiesterase inhibitor. A number of experiments in order to investigate the effect of such an inhibitor, IBMX, are performed, however. As can be expected, the larval
Pharmacological
control
117
of cilia
Table I. Summary of the effects of monoamines and monoamine precursors on the larval content of CAMP c
Ag*ntr 1rryPtoPhan
DO -
L 6b
1 0
INwadr*naline
I,
minutes
Fig. 2. Effects of a phosphodiesterase inhibitor, IBMX, on the larval content of CAMP in the winter spawning species Strongylocentrotus droebachiensis expressed as percentage deviations from a control without IBMX. Salinity 32 promilk. Abscissa: incubation time in minutes. (a) Effects of IBMX (10m4M) at two different stages of development, blastula (65 hr): squares; early gastrula: triangles. (b) Effects
of two concentrations
of IBMX at an early gastrula stage,
lo-’ M: circles; lo-“ M: triangles.
content of CAMP is often greater in the presence of IBMX than in its absence (cf. Fig. 2.). In short experiments with material sampled from different stages of development it is also clear that the IBMX effect depends on the stage concerned, i.e. that a significant increase in CAMP only occurs in stages where the CAMP content shows an increase also in the absence of IBMX. It is also important that maxima as well as minima in CAMP occur in spite of the presence of IBMX 10m4 M which suggests that the diesterase inhibition at this IBMX concentration is only partial. This is supported by the short time experiment in Fig. 2 with an increased concentration of IBMX (1O-3 M). With this concentration the final decrease in CAMP in the control is also counteracted by IBMX. Effects of monoamines and monoamine larval content of CAMP
precursors
on
A great number of experiments are carried out in order to elucidate the effects of monoamines. The incubations are generally performed in the presence of IBMX 10m4 M, a concentration which to some extent protects CAMP from the phosphodiesterase but still allows a decline during periods of low CAMP formation as in Fig. 2.
1o-5
AA
AA
AA
AA
16’
AA
I,
lcTS
AA A
Isopr*nalino
120
-4
10-S
I, ,,
L
10
I,
Oopamino
minutes
Swotonin
60
10e4 -4
10
rywine
lo-’ 10-S
A AA
A
1o-6
10-4
AA AA
AAA-7 (v 'I)
Upward arrowheads indicate stimulation, downward: inhibition. The number of arrowheads refers to the intensity of the effect. A hyphen between arrowheads indicate a change in effect during the developmental phase concerned. Downward arrowheads within brackets refer to inhibition in experiments with high basal content of CAMP. Times of incubatmn: Tryptophan and tyrosine up to 60 min; NA: 30min; DA: 30 min: Serotonin: 40 min; Isoprenaline: 60 min. In most experiments the effects refer to one or a number of samples from cultures in Ga, and Ga, or just one of these periods or in a more advanced stage (prisms). The agents concerned are added to the cultures 60 min before sampling. It is evident from Table 1 that, in short term experiments, a number of monoamines at 10m4 M bring about a strong increase in CAMP formation. This conclusion can be extended to the monoamine precursors 1-tryptophan and 1-tyrosine although, in these cases, the experiments are carried out in the absence of IBMX; furthermore, the early stimulation soon turns into inhibition, presumably due to some feedback mechanism. The difference between the isoprenaline response in Ga, and that in Ga, (Fig. 3) might suggest some phase specificity, but may also reflect an excessive monoamine effect due to intrinsic monoamines (which increase in late Ga,), combined with effects of the apparently very potent isoprenaline. This may quickly lead to feedback inhibition of CAMP formation, maybe also to a strong phosphodiesterase activation; cf. also the variable effects of isoprenaline in Ga, in Table 1. In any case, there is little evidence for a phase specificity in the response to noradrenaline (Fig. 3). The importance of the supposed feedback inhibition is illustrated in a series of experiments with repetitive short interval sampling during the period of incubation, cf. the experiments with noradrenaline 10m4 M, dopamine 10m4M and serotonin lo-’ M (see example in Fig. 4). It should be noted, however, that the decrease in CAMP is only temporary as shown in experiments where sampling continues long enough.
SHERIF SOLIMAN
178
Table 2. Effect of a /I-adrenergic blocker, propranolol (prop) 5 x 10e5 M on the response to noradrenaline (NA). Time of incubation: 10min; preincubation with prop; 30min incubation with prop + NA. Symbols as in Table I
I
NA.'IO-% IO- 5*
vv v
AA
AA
NA’
I
Effects of an adenylcyclase activator on the CAMP response to monoamines
Fig. 3. Effects of isoprenaline and noradrenaline (NA) on the larval content of CAMP in Psammechinus miliaris and Echinus esculentus respectively. Material incubated for 60 and 30 min respectively starting at different developmental stages. Results presented as percentage deviations from a control with IBMX (1O-4 M). Isoprenaline (10m4 M) + IBMX (IO-“ M): circles; NA (10m4 M) + IBMX (1O-4 M): triangles.
The activity of adenylcyclase involves a cooperation between two proteins. One is catalytic; the other activates the catalytic unit, a function requiring the presence of GTP or a more stable structural analogue, e.g. p(NH)ppG (cf. Ross and Gilman, 1980). According to Soliman (1984) an admixture of p(NH)ppG to solutions of various monoamines enhances their stimulatory effect on ciliary activity. If the combined effects are too strong, however, ciliary inhibition occurs. The ciliary effects are closely reminiscent of the present ones where the CAMP content is used as the parameter. The initial increase in CAMP is somewhat attenuated in the presence of p(NH)ppG which may reflect a decrease in the affinity of the receptor to the agonist (cf. Ross and Gilman, 1980), but the so-called
The effect of noradrenaline is considerable also if the agent is added to homogenates of young plutei. Effect of proprunolol on the effect of norudrenaline
I
Gal
+100
In an earlier paper it was shown that the effect of noradrenaline is mediated by a- as well as by /I-receptors (Soliman, 1983b). It is therefore of interest to study the effects of noradrenaline antagonists of different specificity on the noradrenaline induced formation of CAMP. In the presence of IBMX (10m4 M) propranolol (5 x 10e5 M) strongly counteracts the noradrenaline effect and even brings about a decrease below the control level (Table 2).
,~, 120min
60min
: ‘. ,:’
t
KS’1
Sea water: IOrnM
+2oc
4oc
120 min
60 min +101
.’
‘\,
c-
. . . . .
c--- .
,j
I *.
0 25
50
301
/‘““l’
rJ .,.,,
”
‘.. xc=”
75
Fig. 4. Elfects of the adenylcyclase stimulator P(NH)ppG on the response to NA, presented as a percentage deviations from a control with IBMX (10m4 M) in larvae of Psatnmechinus miliuris in Ga,. Salinity 32 promille. NA line; NA solid (1O-4 M) + IBMX (1O-4 M): (1O-4 M) + IBMX (lo-” M) + P(NH)ppG (10m6 M): dashed line.
Fig. 5. Effect of increased calcium concentration of the medium on the larval content of CAMP and cGMP in Psammechinus miliaris. Salinity 26 promille. Upper part: effects in Ga,; Lower part: effects in Ga,. Solid line corresponds to CAMP, dotted line to cGMP. Results presented as percentage deviations from a control. No IBMX is used in this experiment.
179
Pharmacological control of cilia feedback inhibition may be cancelled (Fig. 4). With higher concentrations of p(NH)ppG, on the other hand, the feedback inhibition becomes pronounced. Efects of altered calcium concentration content of CAMP and cGMP
p + 50
Ga2
3
on the larval
Although the function of the activator unit of adenylcyclase is Ca2+ dependent, the catalytic unit is inhibited by increased concentrations of Ca2+, [Ca’+] (cf. Mahafee and Ontjes, 1980 and Rassmussen, 1981). The effects of altered [Ca’+] of the sea water on ciliary activity shows some regularities that may be related to this (cf. Soliman, 1984). In brief, an increase in [Ca2+] may bring about a very brief initial stimulation, and the Ga, effects of the Ca2+ ionophore A 23187 are stimulatory as well. Stimulation in these cases could reflect the role of Ca2+ as a charge carrying ion in ciliary excitation and/or its positive role for the activator protein. Quite soon, however, inhibitory effects prevail. A lowered [Ca’+], on the other hand, down to a certain limit, brings about an increased ciliary activity. These effects might reflect Ca2+ dependent variations in the activity of the catalytic unit. The present study indicates that at least some of the inhibitory and stimulatory effects of altered [Ca2+] do reflect variations in the CAMP content in the expected directions. With just one exception, increased [Ca*+] per se thus lowers the CAMP content (Fig. 5). Also the stimulatory effects of a monoamine (NA, 10m4M) on CAMP accumulation is inhibited by 80% in Ca2+ enriched sea water (16 mM Ca2+). Furthermore, a moderately lowered [Ca’+] brings about an increase in the level of CAMP (Fig. 6). One should consider, however, that the phosphodiesterase
E E e 6 # k c) 0 -50
-100 Fig. 7. Effects of muscarinic agents (10m4M) on the content of cGMP in larvae of Psammechinus miliaris. Salinity 26 promille. Time of incubation: 60 min. Results presented as percentage deviations from a control in sea water without IBMX. activity is also Ca 2+ dependent. The results are in line with the ciliary effects of lowered [Ca’+] and suggest that Ca2+ may play a positive role for adenylcyclase activity separate from the negative one (Soliman, 1983~). The activity of guanylcyclase is positively dependent on Ca2+. This is in line with the effect of increased [Ca*+] on the cGMP content of the larvae as shown in Fig. 5. Effects of muscarinic
6
hours
Fig. 6. Effects of a decreased calcium concentration on the content of CAMP in larvae of Psammechinus miliaris. Salinity 21 promille. Results presented as percentage deviations from a control. Arrow: as in Fig. 1. No IBMX is used in the experiment.
agents on the cGMP
content
The effects of a number of muscarinic agents on the larval content of cGMP in Psammechinus miliaris (salinity 26 promille) is shown in Fig. 7. It is quite clear that oxotremorine, a purely muscarinic agent, and metacholine, where the muscarinic activity predominates, both bring about a considerable decrease in the larval content of cGMP, in Ga, as well as in Ga,. Acetylcholine and carbachol, which besides their strong nicotinic activities have strong muscarinic effects, also reduce cGMP. The results presented appear paradoxical as muscarinic effects are often linked to guanylcyclase activation. The primary target of the agonists concerned, however, may be restricted to a superficial cell membrane linked guanylcyclase which may only represent a small fraction of the total cellular enzyme pool, i.e. most of the cyclase activity may be due to a soluble nonmembrane linked component directly stimulated by Ca2+ entering the cells. If the membrane linked cGMP production lowers the cellular influx of Ca2+, as opposed to CAMP, the results described become comprehensible. A muscarinic activation of membrane linked guanylcyclase may thus decrease the influx of Ca2+ leading to a reduction in the main, soluble Ca2+ activated component. The results described are to a great extent in line with the suggestion that the nicotinic increase in Ca2+ influx is modulated by muscarines (cf. Soliman, 1983a).
180
SERIF S~LIMAN
It may be significant that the spontaneous accumulation of cGMP in IBMX treated larvae is significant only in Ga, which is in line with the increase in the larval acetylcholine content at this stage of development (cf. Buznikov et al., 1968). However, in experiments with frequent sampling in control cultures, fluctuations in cGMP also occur during Ga, opposite to the maxima and minima in CAMP (Fig. 2, experiment without IBMX). In this case the Ga, cGMP peak may reflect a Ca2+ influx induced by CAMP (see the Discussion section). One might therefore expect that nicotine treatment in Ga, also brings about an increase in cGMP, if such an effect is not counteracted by the cell surface effect of the cGMP formed. Much more work is required in order to elucidate this type of feedback interactions between different compartments of guanylcyclase. Effects of muscarinic CAMP
agents on the larval content of
As a supplement to the studies on the role of Ca2+ in the regulation of ciliary activity and the level of cyclical nucleotides, some experiments on the effects of muscarinic agents on the CAMP level are carried out since muscarinic agents may be expected to affect, indirectly, the adenylcyclase activity. As in the preceeding section, effects of agents with pure muscarinic activity are used as well as such that, in addition, are strongly nicotinic. Although the results of a considerable number of experiments are available, the conclusions may still be considered preliminary, in particular as the effects of Ca2+ on the adenylcyclase complex may be positive as well as negative. The opposite roles of Ca 2+ for the two adenylcyclase units may, anyhow, facilitate the discussion. Oxotremorine, a purely muscarinic agent, which according to the previous section may reduce the influx of Ca2+, at 10m4 M brings about a considerable increase in the larval content of CAMP after 60 min
G=2
1
Fig. 8. Effects of a muscarininc
agent, oxotremorine
(10m4 M) on the content of cAMPin larvae of Psammechinus miliuris. Salinity 26 promille. Results presented as percentage deviations from a control with IBMX (10e4M). Unfilled blocks correspond to 60 min of incubation, dashed blocks to 120 minutes.
of incubation at the salinity 26 promille (Fig. S), in Ga, as well as in Ga,. Upon prolonged incubation, however, inhibition occurs. Temporary stimulatory effects have been observed in experiments with arecoline ( 10e4 M) and carbachol(l0 -4 and 10 5M) but only in Ga, The temporary stimulatory and effect of arecoline and carbachol in Ga, are paralleled by the experiments with acetylcholine at the same concentration. DISCUSSION
The present results of chemical studies on the developmental changes in the larval content of CAMP and cGMP and of the effects of different agonists and altered [Ca’+] in the medium are, to a great extent, in agreement with suggestions expressed on the basis of preceding investigations (Soliman, 1983a, 1983b, 1984). Thus, the developmental peaks in CAMP and cGMP have a clear relation to the two main phases of gastrulation and ciliary activity (cf. Fig. 1.) Also the minimum in the amount of cyclic nucleotides at the Ga,-Ga, border support the idea that CAMP and cGMP are involved in a control system shared by the morphogenetic cell movements and ciliary activity. The variation in the larval content of the two cyclic nucleotides presumably reflect fundamental developmental changes in the pharmacological signal system. Thus, in light of the earlier experiments on the pharmacological control of ciliary activity (Soliman, 1983a) and the morphogenetic studies by Gustafson and Toneby (1970, 1971) together with the analytical results of Buznikov et al. (1964, 1968) the Ga, as well as the “post-hatching” peak of CAMP can be considered as a result of high monoamine production. The CAMP minimum at the Ga,-Ga, border may reflect a decrease in monoamine synthesis but also a negative feedback effect on CAMP caused by high Ca2+ influx through the cell membrane, due to the proceding high CAMP formation, which may hamper the activity of adenylcyclase. As has been suggested the Ga, peak during a period of low monoamine formation, on the other hand, could be an indirect result of an increased muscarinic activity due to acetylcholine which, via cGMP, reduces the Ca2+ influx, this leading to a lowered repression of adenylcyclase activity. The nicotinic effect of acetylcholine may also play a role. The final increase in CAMP seen in Fig. 1 may reflect a renewed increase in monoamine formation. This explanation of the developmental changes in cyclical nucleotides, speculative as it may be, is supported by the present observations on monoaminergic and cholinergic effects on the larval content of CAMP and also by the effects of an altered Ca2+ concentration of the medium. The developmental changes in cGMP fit with this model. Thus, the Ga, peak in cGMP is presumably the result of an increased Ca2+ influx induced by CAMP. The Ga, peak, on the other hand, may be caused by an increased acetylcholine formation (cf. Buznikov et al., 1968). The effects of muscarinic agents on the cGMP contents, however, are not consistent with this model. As has been suggested in the Results section, however, the effects of added muscarines on cGMP may be explained by the occurrence of two fractions
Pharmacological control of cilia of guanylcyclase, a minor one bound to the cell surface and controlling the Ca*+ influx, a major one of endocellular location and indirectly activated by the Ca*+ influx into the cell. The suggested interaction between the two cyclase systems, the feedback inhibition of the adenylcyclase, and the suggested interplay between a cell membrane linked and an endocellular pool of guanylcyclase, all presumably related to the cellular content of ionic Ca*+, an ion that presumably also plays a direct role in cellular excitation, may still be considered a working hypothesis.
Acknowledgements-This paper is dedicated to my teacher, Professor Tryggve Gustafson, to whom I would like to express my cordial thanks for encouragement, stimulating discussions and valuable criticism during the experimental studies and the preparation of the manuscript. Many thanks are directed to Professor Jarl-Ove Stromberg and the staff of Kristineberg’s Marine Biological Station for working facilities and supply of material. The skilful assistance by Mrs Anna-Lena Kullgren and the technical advice by Miss Marie-Louise Tjornhammar is gratefully acknowledged. I am also grateful to Mrs Renate Ahlfont for typing the manuscript. This project has been supported by a grant from the Swedish Natural Science Research Council to Tryggve Gustafson. REFERENCES Brown
B. L., Albano J. D. M., Ekins R. P. and Scherzi A. M. (1971) A simple and sensitive saturation assay method for the measurement of adenosine 3’5’-cyclic monophosphate. Biochem. J. 21, 561-562. Buznikov G. A., Chudakova 1. V. and Zvezdina N. D. (1964) The role of neurohumours in early embryogenesis-I. Serotonin content of developing embryos of sea urchin and loach. J. Embryol. exp. Morphol. 12, 563-573.
Buznikov G. A., Chudakova I. V., Berdysheva L. V. and Vyazmina N. M. (1968) The role of neurohumours in
181
early embryogenesis-II. Acetycholine and catecholamine content in developing embryos of sea urchins. J. Embryol. exp. Morphol. 20, 119-128. Gustafson T. (1981) The cellular basis of morphogenesis and behaviour during sea urchin development. In The Biology of Normal Human Growth (Edited by Ritzen M.), pp. 1299137, Raven Press, New York. Gustafson T. and Toneby M. (1970) The role of serotonin and acetylcholine in sea urchin morphogenesis. Exp. Cell Res. 62, 102-117. Gustafson T. and Toneby M. (1971) How genes control morphogenesis. Am. Sci. 59, 452462. Gustafson T. and Treufeldt R. (1981) Neuropharmacological analysis of mechanisms controlling larval behaviour in the sea urchin-I. Effects of carbamylcholine and acetylcholine. Acta Zool. 62, 233-249. Mahafee D. D. and Ontjes D. A. (1980) The role of calcium in the control of adrenal adenylate cyclase. Enhancement of enzyme activation by guanyl-5’-yl-imidodiphosphate. J. biol. Chem. 255, 1565-1571. Rasmussen H. (1981) Calcium and CAMP as Synarchic Messengers. John Wiley, New York. Ross E. M. and Gilman A. G. (1980) Biochemical properties of hormone stimulated adenylate cyclase. A. Rev. Biothem. 49, 533-564.
Solimans S. (1983a) Pharmacological control of ciliary activity in the young sea urchin larva. Effects of cholinergic agonists and antagonists. Comp. Biochem. Physiol. 74c,
397-407.
Soliman S. (1983b) Pharmacological control of ciliary activity in the young sea urchin larva. Effects of monoaminergic agents. Comp. Biochem. Physiol. 76C, 18 l-1 9 I. Soliman S. (1984) Pharmacological control of ciliary activity in the young sea urchin larva. Studies on the role of calcium and cyclic nucleotides. Camp. Biochem. Physiol. 78C, 183-191. Toneby M. (1980) Dopamine in developing larvae of the sea urchin Psammechinus miliaris. Comp. Biochem. Physiol. 65C, 139-142.
Treufeldt R. and Gustafson T. (1981) Neuropharmacological analysis of mechanisms controlling larval behaviour in the sea urchin-II. Effect of physostigmine and the role of cholinergic mechanism. Acta. 2001. 62, 249-258.
Biochem.
Printed in Great
Phvsiol.
Vol. 78C, No. I, pp. 175-181, 1984 c
B&in
0306-4492/84 $3.00 + 0.00 1984 Pergamon Press Ltd
PHARMACOLOGICAL CONTROL OF CILIARY ACTIVITY IN THE YOUNG SEA URCHIN LARVA: CHEMICAL STUDIES ON THE ROLE OF CYCLIC NUCLEOTIDES SHERIF S~LIMAN The Wenner-Gren
Institute
for Experimental Biology, University 45 Stockholm, Sweden. Telephone:
of Stockholm, 08-34-0860
Norrtullsgatan
16, S-l 13
(Received 29 June 1983) Abstract-l. Distinct peaks in CAMP and cGMP content during early development, partly opposite to each other, may be correlated with the two main phases of gastrulation and ciliary activity. 2. Monoamines increases CAMP formation. A transient or extended decrease follows, presumably reflecting some feedback mechanism. Muscarinic agents and CaZ+ interfere. 3. The developmental variation in cyclic nucleotides may reflect a temporal shift in the role of various signal substances as well as feedback regulation related to Ca2+ influx. 4. The opposite changes in CAMP and cGMP during early gastrulation may reflect a mutual dependency of the two nucleotide cyclases related to changes in Ca2+ influx.
INTRODUCTION
morphogenetic cell activities are both closely related to the cellular level of ionic calcium in some important compartments. The results can also be considered as a supplement to the studies on the neuropharmacological control of muscular activity in the pluteus larva carried out by Gustafson and Treufeldt (1981) and Treufeldt and Gustafson (1981) and may hence contribute to the elucidation of the problem of ontogeny of behaviour as ciliary activity starts before muscular activity, although the control mechanisms may be partly the same.
In earlier papers in this series, the control of larval ciliary activity was approached by studies on the ciliary effects of cholinergic and monoaminergic agonists and antagonists (Soliman, 1983a,b). As a further step in the analysis, the effects of cyclic nucleotides and calcium ions in connection with ciliary activity was investigated since they may be presumed to mediate the agonist effects concerned (Soliman, 1984). In this paper the results of chemical analyses of cyclical nucleotides are documented, both with regard to developmental changes in normal larvae and to the effects of various agonists and alterations of the calcium content of the sea water. Although some of the results of the present study may still be regarded as preliminary, in particular with respect to some changes in cGMP, the observations support the idea that calcium is involved in ciliary regulation and that the cyclic nucleotides have a central role in the membrane flux of calcium ions and vice versa, i.e. that cyclic nucleotides and calcium ions have a mutual relationship as in many other systems more extensively studied (cf. Rasmussen, 1981). The results also contribute to the discussion on the so-called phase specificity of different physiological signal substances in early development (cf. Gustafson and Toneby, 1970, 1971 and Gustafson and Treufeldt, 1981), and may shed some light on the mechanism behind the often drastic fluctuation in the response to agonists as development proceeds, a phenomenon that may overshadow their physiological phase specificity (cf. Soliman, 1983a, 1983b). The final goal of the investigation is to shed some light on the morphogenetic cell activities during gastrulation which attend the start of the ciliary activity and its further changes to a maximum, a minimum and a renewed peak. In fact, it is attractive to think that the ciliary control and the control of
MATERIALS AND METHODS Young sea urchin larvae were used, from hatching, up to an early prism stage, and in some experiments, plutei. Most experiments are carried out with the summer spawning species Psammechinus miliaris, the shallow and the deep water forms living at a salinity of 21 or 26 promille and at 32 promille respectively. Experiments with the winter spawning species Strongylocentrotus droebachiensis and Echinus esculentus, after capture reared at a salinity of 32 promille, are also carried out. After artificial fertilization the material is cultured in beakers under gentle stirring at constant temperatures, around 16” and 8°C respectively. The culture density varies between 500 and 1500 larvae per ml depending on which cyclic nucleotide is to be analyzed, the determination of cGMP requiring a higher larval concentration than that of CAMP. The salinity of the sea water used for the cultures is exactly adjusted to 21, 26 or 32 promille depending on the source of the material. Studies on the effects of altered calcium concentration of the medium are performed by admixture of calcium free and calcium enriched artificial sea water to the normal sea water, but rearing of the larvae, up to the start of the experiments, is always carried out in normal sea water. The effects of various agents on the larval content of cyclical nucleotides are investigated by adding the agent concerned, dissolved in sea water followed by pH adjustment, to the larval suspension. The length of incubation varies between the experiments and is defined in the figure 175
176
SHERIF SOLIMAN
captions. The phosphodiesterase inhibitor 3-isobutyl-lmethylxanthine (IBMX) IO-” M is added to the incubation medium unless otherwise stated. Incubation is quickly terminated by rapid hand centrifugation of the culture sample after which the supematant is replaced by chilled extraction medium (0.3 M perchloric acid, 1 mM EDTA and 10% of acidic methanol 0.1 M HCl in methanol). The larvae are homogenized in a Dounce homogenizer in the extraction medium after which the homogenate is kept for 1 hr during continued chilling in an ice bath. Extraction is terminated by centrifugation at 6000 rpm for 10 min after which the supernatant is transferred into another centrifuge tube where perchloric acid is precipitated by adding 8&85~1 of KOH per ml. After centrifugation at 3000rpm for 10min the supematant is collected in a test tube where pH is adjusted to 5-6 by adding 10 ~1 concentrated acetic acid. A volume of 5&100 pl of the final extract is used for the
determination of the cyclic nucleotides. CAMP is assayed according to the method of Brown et al. (1971) or by using a CAMP assay kit from Radiochemical Centre, Amersham, U.K. cGMP is assayed by aid of the Amersham cGMP radioimmunoassay kit.
RESULTS
Developmentul and cGMP
changes in the larval content of CAMP
A considerable number of experiments on the normal developmental changes in the larval content of cyclic nucleotides are carried out, in particular with respect to CAMP which is studied both in warm and cold water spawning species. The experiments are carried out without IBMX preincubation. The cGMP content turns out to be much lower than that of CAMP (cf. Fig. l), and the number of larvae in each culture sample is adjusted to this.
I
Id’pmol
cGMP/103
pmol cAMP/103
larvae
larvae
I8 20 22 24 24 Time after fertlllzotlon
t
28 (hr)
Fig. 1, Changes in content of CAMP and cGMP during early larval development of Psammechinus miliaris. Salinity 26 promille. Arrow indicates the border between Ga, and Ga,. Note that the CAMP content is lOO-fold higher than that of cGMP. The points correspond to mean + standard error of mean of duplicate determinations in 8-15 cultures. CAMP: solid line: cGMP: dotted line.
The changes in CAMP in the summer spawning species Psammechinus miliaris are quite reproducible. A CAMP peak is seen during Ga, , a minimum occurs at the Ga,-Ga, border, and a renewed increase is seen in Ga, leading to a peak (Fig. 1) or to a plateau. In the advanced gastrula, close to a prism stage, the CAMP content shows a steep rise. When sampling starts somewhat later, the decline after this early peak is clear (Fig. 1). There is evidently a striking parallelism between the temporal variation in the larval content of CAMP here described and the developmental changes in ciliary activity described by Soliman (1983a) in the sense that in both cases a Ga, maximum, a minimum around the Ga,-Ga, border and a new peak or at least a rise can be seen. The only difference is the early “post-hatching peak” in CAMP which is not seen in the assay of ciliary activity; it should be mentioned, however, that in certain experiments with neuropharmaca the initial ciliary activity in the controls is fairly high (cf. Soliman, 1984). It should also be noted that the assays of ciliary activity are interrupted in the advanced gastrula more or less before the final steep increase in CAMP since, at this stage, the larvae change their external shape and their pattern of swimming (see Soliman, 1983a). A general parallelism between the larval content of monoamines and CAMP ciliary activity can be noted (cf. Buznikov et al., 1964 and Soliman, 1983a). This parallelism is only lacking during early Ga, where the monoamine content is low, whereas the CAMP content shows an increase, to a peak or a plateau. This suggest that the formation of CAMP not only reflects the fluctuations in the larval content of monoamines but that variation in CAMP and hence ciliary activity is also governed by some kind of temporary, negative feedback regulation. One should also consider that, as gastrulation proceeds, the role of cholinergic signals may increase (cf. Buznikov et al., 1968) which may also affect the adenylcyclase activity, as discussed below. The Ga, changes in the larval content of cGMP are to a great extent opposite to those in CAMP (Fig. 1). In Ga,, on the other hand, the two cyclic nucleotides show a rather coordinated rise. The Ga, pattern suggests a mutual dependency of the two nucleotide cyclases, e.g. a strong influx of Ca*+ induced by CAMP may hamper the adenylcyclase activity whereas it promotes the guanylcyclase which, via cGMP, hampers the influx of Ca*+ and therefore promotes the adenylcyclase, etc. In Ga, one may presume that the increase in acetylcholine (cf. Buznikov et al., 1968) brings about a muscarinic activation of the guanylcyclase which still may have a positive effect on the adenylcyclase (cf. the preceding paragraph). At the end of Ga,, the inverse relationship may appear again although the experiments are fragmentary. Effects of a phosphodiesterase content of CAMP
inhibitor on the larval
The results presented in the previous section are carried out in the absence of a phosphodiesterase inhibitor. A number of experiments in order to investigate the effect of such an inhibitor, IBMX, are performed, however. As can be expected, the larval
Pharmacological
control
117
of cilia
Table I. Summary of the effects of monoamines and monoamine precursors on the larval content of CAMP c
Ag*ntr 1rryPtoPhan
DO -
L 6b
1 0
INwadr*naline
I,
minutes
Fig. 2. Effects of a phosphodiesterase inhibitor, IBMX, on the larval content of CAMP in the winter spawning species Strongylocentrotus droebachiensis expressed as percentage deviations from a control without IBMX. Salinity 32 promilk. Abscissa: incubation time in minutes. (a) Effects of IBMX (10m4M) at two different stages of development, blastula (65 hr): squares; early gastrula: triangles. (b) Effects
of two concentrations
of IBMX at an early gastrula stage,
lo-’ M: circles; lo-“ M: triangles.
content of CAMP is often greater in the presence of IBMX than in its absence (cf. Fig. 2.). In short experiments with material sampled from different stages of development it is also clear that the IBMX effect depends on the stage concerned, i.e. that a significant increase in CAMP only occurs in stages where the CAMP content shows an increase also in the absence of IBMX. It is also important that maxima as well as minima in CAMP occur in spite of the presence of IBMX 10m4 M which suggests that the diesterase inhibition at this IBMX concentration is only partial. This is supported by the short time experiment in Fig. 2 with an increased concentration of IBMX (1O-3 M). With this concentration the final decrease in CAMP in the control is also counteracted by IBMX. Effects of monoamines and monoamine larval content of CAMP
precursors
on
A great number of experiments are carried out in order to elucidate the effects of monoamines. The incubations are generally performed in the presence of IBMX 10m4 M, a concentration which to some extent protects CAMP from the phosphodiesterase but still allows a decline during periods of low CAMP formation as in Fig. 2.
1o-5
AA
AA
AA
AA
16’
AA
I,
lcTS
AA A
Isopr*nalino
120
-4
10-S
I, ,,
L
10
I,
Oopamino
minutes
Swotonin
60
10e4 -4
10
rywine
lo-’ 10-S
A AA
A
1o-6
10-4
AA AA
AAA-7 (v 'I)
Upward arrowheads indicate stimulation, downward: inhibition. The number of arrowheads refers to the intensity of the effect. A hyphen between arrowheads indicate a change in effect during the developmental phase concerned. Downward arrowheads within brackets refer to inhibition in experiments with high basal content of CAMP. Times of incubatmn: Tryptophan and tyrosine up to 60 min; NA: 30min; DA: 30 min: Serotonin: 40 min; Isoprenaline: 60 min. In most experiments the effects refer to one or a number of samples from cultures in Ga, and Ga, or just one of these periods or in a more advanced stage (prisms). The agents concerned are added to the cultures 60 min before sampling. It is evident from Table 1 that, in short term experiments, a number of monoamines at 10m4 M bring about a strong increase in CAMP formation. This conclusion can be extended to the monoamine precursors 1-tryptophan and 1-tyrosine although, in these cases, the experiments are carried out in the absence of IBMX; furthermore, the early stimulation soon turns into inhibition, presumably due to some feedback mechanism. The difference between the isoprenaline response in Ga, and that in Ga, (Fig. 3) might suggest some phase specificity, but may also reflect an excessive monoamine effect due to intrinsic monoamines (which increase in late Ga,), combined with effects of the apparently very potent isoprenaline. This may quickly lead to feedback inhibition of CAMP formation, maybe also to a strong phosphodiesterase activation; cf. also the variable effects of isoprenaline in Ga, in Table 1. In any case, there is little evidence for a phase specificity in the response to noradrenaline (Fig. 3). The importance of the supposed feedback inhibition is illustrated in a series of experiments with repetitive short interval sampling during the period of incubation, cf. the experiments with noradrenaline 10m4 M, dopamine 10m4M and serotonin lo-’ M (see example in Fig. 4). It should be noted, however, that the decrease in CAMP is only temporary as shown in experiments where sampling continues long enough.
SHERIF SOLIMAN
178
Table 2. Effect of a /I-adrenergic blocker, propranolol (prop) 5 x 10e5 M on the response to noradrenaline (NA). Time of incubation: 10min; preincubation with prop; 30min incubation with prop + NA. Symbols as in Table I
I
NA.'IO-% IO- 5*
vv v
AA
AA
NA’
I
Effects of an adenylcyclase activator on the CAMP response to monoamines
Fig. 3. Effects of isoprenaline and noradrenaline (NA) on the larval content of CAMP in Psammechinus miliaris and Echinus esculentus respectively. Material incubated for 60 and 30 min respectively starting at different developmental stages. Results presented as percentage deviations from a control with IBMX (1O-4 M). Isoprenaline (10m4 M) + IBMX (IO-“ M): circles; NA (10m4 M) + IBMX (1O-4 M): triangles.
The activity of adenylcyclase involves a cooperation between two proteins. One is catalytic; the other activates the catalytic unit, a function requiring the presence of GTP or a more stable structural analogue, e.g. p(NH)ppG (cf. Ross and Gilman, 1980). According to Soliman (1984) an admixture of p(NH)ppG to solutions of various monoamines enhances their stimulatory effect on ciliary activity. If the combined effects are too strong, however, ciliary inhibition occurs. The ciliary effects are closely reminiscent of the present ones where the CAMP content is used as the parameter. The initial increase in CAMP is somewhat attenuated in the presence of p(NH)ppG which may reflect a decrease in the affinity of the receptor to the agonist (cf. Ross and Gilman, 1980), but the so-called
The effect of noradrenaline is considerable also if the agent is added to homogenates of young plutei. Effect of proprunolol on the effect of norudrenaline
I
Gal
+100
In an earlier paper it was shown that the effect of noradrenaline is mediated by a- as well as by /I-receptors (Soliman, 1983b). It is therefore of interest to study the effects of noradrenaline antagonists of different specificity on the noradrenaline induced formation of CAMP. In the presence of IBMX (10m4 M) propranolol (5 x 10e5 M) strongly counteracts the noradrenaline effect and even brings about a decrease below the control level (Table 2).
,~, 120min
60min
: ‘. ,:’
t
KS’1
Sea water: IOrnM
+2oc
4oc
120 min
60 min +101
.’
‘\,
c-
. . . . .
c--- .
,j
I *.
0 25
50
301
/‘““l’
rJ .,.,,
”
‘.. xc=”
75
Fig. 4. Elfects of the adenylcyclase stimulator P(NH)ppG on the response to NA, presented as a percentage deviations from a control with IBMX (10m4 M) in larvae of Psatnmechinus miliuris in Ga,. Salinity 32 promille. NA line; NA solid (1O-4 M) + IBMX (1O-4 M): (1O-4 M) + IBMX (lo-” M) + P(NH)ppG (10m6 M): dashed line.
Fig. 5. Effect of increased calcium concentration of the medium on the larval content of CAMP and cGMP in Psammechinus miliaris. Salinity 26 promille. Upper part: effects in Ga,; Lower part: effects in Ga,. Solid line corresponds to CAMP, dotted line to cGMP. Results presented as percentage deviations from a control. No IBMX is used in this experiment.
179
Pharmacological control of cilia feedback inhibition may be cancelled (Fig. 4). With higher concentrations of p(NH)ppG, on the other hand, the feedback inhibition becomes pronounced. Efects of altered calcium concentration content of CAMP and cGMP
p + 50
Ga2
3
on the larval
Although the function of the activator unit of adenylcyclase is Ca2+ dependent, the catalytic unit is inhibited by increased concentrations of Ca2+, [Ca’+] (cf. Mahafee and Ontjes, 1980 and Rassmussen, 1981). The effects of altered [Ca’+] of the sea water on ciliary activity shows some regularities that may be related to this (cf. Soliman, 1984). In brief, an increase in [Ca2+] may bring about a very brief initial stimulation, and the Ga, effects of the Ca2+ ionophore A 23187 are stimulatory as well. Stimulation in these cases could reflect the role of Ca2+ as a charge carrying ion in ciliary excitation and/or its positive role for the activator protein. Quite soon, however, inhibitory effects prevail. A lowered [Ca’+], on the other hand, down to a certain limit, brings about an increased ciliary activity. These effects might reflect Ca2+ dependent variations in the activity of the catalytic unit. The present study indicates that at least some of the inhibitory and stimulatory effects of altered [Ca2+] do reflect variations in the CAMP content in the expected directions. With just one exception, increased [Ca*+] per se thus lowers the CAMP content (Fig. 5). Also the stimulatory effects of a monoamine (NA, 10m4M) on CAMP accumulation is inhibited by 80% in Ca2+ enriched sea water (16 mM Ca2+). Furthermore, a moderately lowered [Ca’+] brings about an increase in the level of CAMP (Fig. 6). One should consider, however, that the phosphodiesterase
E E e 6 # k c) 0 -50
-100 Fig. 7. Effects of muscarinic agents (10m4M) on the content of cGMP in larvae of Psammechinus miliaris. Salinity 26 promille. Time of incubation: 60 min. Results presented as percentage deviations from a control in sea water without IBMX. activity is also Ca 2+ dependent. The results are in line with the ciliary effects of lowered [Ca’+] and suggest that Ca2+ may play a positive role for adenylcyclase activity separate from the negative one (Soliman, 1983~). The activity of guanylcyclase is positively dependent on Ca2+. This is in line with the effect of increased [Ca*+] on the cGMP content of the larvae as shown in Fig. 5. Effects of muscarinic
6
hours
Fig. 6. Effects of a decreased calcium concentration on the content of CAMP in larvae of Psammechinus miliaris. Salinity 21 promille. Results presented as percentage deviations from a control. Arrow: as in Fig. 1. No IBMX is used in the experiment.
agents on the cGMP
content
The effects of a number of muscarinic agents on the larval content of cGMP in Psammechinus miliaris (salinity 26 promille) is shown in Fig. 7. It is quite clear that oxotremorine, a purely muscarinic agent, and metacholine, where the muscarinic activity predominates, both bring about a considerable decrease in the larval content of cGMP, in Ga, as well as in Ga,. Acetylcholine and carbachol, which besides their strong nicotinic activities have strong muscarinic effects, also reduce cGMP. The results presented appear paradoxical as muscarinic effects are often linked to guanylcyclase activation. The primary target of the agonists concerned, however, may be restricted to a superficial cell membrane linked guanylcyclase which may only represent a small fraction of the total cellular enzyme pool, i.e. most of the cyclase activity may be due to a soluble nonmembrane linked component directly stimulated by Ca2+ entering the cells. If the membrane linked cGMP production lowers the cellular influx of Ca2+, as opposed to CAMP, the results described become comprehensible. A muscarinic activation of membrane linked guanylcyclase may thus decrease the influx of Ca2+ leading to a reduction in the main, soluble Ca2+ activated component. The results described are to a great extent in line with the suggestion that the nicotinic increase in Ca2+ influx is modulated by muscarines (cf. Soliman, 1983a).
180
SERIF S~LIMAN
It may be significant that the spontaneous accumulation of cGMP in IBMX treated larvae is significant only in Ga, which is in line with the increase in the larval acetylcholine content at this stage of development (cf. Buznikov et al., 1968). However, in experiments with frequent sampling in control cultures, fluctuations in cGMP also occur during Ga, opposite to the maxima and minima in CAMP (Fig. 2, experiment without IBMX). In this case the Ga, cGMP peak may reflect a Ca2+ influx induced by CAMP (see the Discussion section). One might therefore expect that nicotine treatment in Ga, also brings about an increase in cGMP, if such an effect is not counteracted by the cell surface effect of the cGMP formed. Much more work is required in order to elucidate this type of feedback interactions between different compartments of guanylcyclase. Effects of muscarinic CAMP
agents on the larval content of
As a supplement to the studies on the role of Ca2+ in the regulation of ciliary activity and the level of cyclical nucleotides, some experiments on the effects of muscarinic agents on the CAMP level are carried out since muscarinic agents may be expected to affect, indirectly, the adenylcyclase activity. As in the preceeding section, effects of agents with pure muscarinic activity are used as well as such that, in addition, are strongly nicotinic. Although the results of a considerable number of experiments are available, the conclusions may still be considered preliminary, in particular as the effects of Ca2+ on the adenylcyclase complex may be positive as well as negative. The opposite roles of Ca 2+ for the two adenylcyclase units may, anyhow, facilitate the discussion. Oxotremorine, a purely muscarinic agent, which according to the previous section may reduce the influx of Ca2+, at 10m4 M brings about a considerable increase in the larval content of CAMP after 60 min
G=2
1
Fig. 8. Effects of a muscarininc
agent, oxotremorine
(10m4 M) on the content of cAMPin larvae of Psammechinus miliuris. Salinity 26 promille. Results presented as percentage deviations from a control with IBMX (10e4M). Unfilled blocks correspond to 60 min of incubation, dashed blocks to 120 minutes.
of incubation at the salinity 26 promille (Fig. S), in Ga, as well as in Ga,. Upon prolonged incubation, however, inhibition occurs. Temporary stimulatory effects have been observed in experiments with arecoline ( 10e4 M) and carbachol(l0 -4 and 10 5M) but only in Ga, The temporary stimulatory and effect of arecoline and carbachol in Ga, are paralleled by the experiments with acetylcholine at the same concentration. DISCUSSION
The present results of chemical studies on the developmental changes in the larval content of CAMP and cGMP and of the effects of different agonists and altered [Ca’+] in the medium are, to a great extent, in agreement with suggestions expressed on the basis of preceding investigations (Soliman, 1983a, 1983b, 1984). Thus, the developmental peaks in CAMP and cGMP have a clear relation to the two main phases of gastrulation and ciliary activity (cf. Fig. 1.) Also the minimum in the amount of cyclic nucleotides at the Ga,-Ga, border support the idea that CAMP and cGMP are involved in a control system shared by the morphogenetic cell movements and ciliary activity. The variation in the larval content of the two cyclic nucleotides presumably reflect fundamental developmental changes in the pharmacological signal system. Thus, in light of the earlier experiments on the pharmacological control of ciliary activity (Soliman, 1983a) and the morphogenetic studies by Gustafson and Toneby (1970, 1971) together with the analytical results of Buznikov et al. (1964, 1968) the Ga, as well as the “post-hatching” peak of CAMP can be considered as a result of high monoamine production. The CAMP minimum at the Ga,-Ga, border may reflect a decrease in monoamine synthesis but also a negative feedback effect on CAMP caused by high Ca2+ influx through the cell membrane, due to the proceding high CAMP formation, which may hamper the activity of adenylcyclase. As has been suggested the Ga, peak during a period of low monoamine formation, on the other hand, could be an indirect result of an increased muscarinic activity due to acetylcholine which, via cGMP, reduces the Ca2+ influx, this leading to a lowered repression of adenylcyclase activity. The nicotinic effect of acetylcholine may also play a role. The final increase in CAMP seen in Fig. 1 may reflect a renewed increase in monoamine formation. This explanation of the developmental changes in cyclical nucleotides, speculative as it may be, is supported by the present observations on monoaminergic and cholinergic effects on the larval content of CAMP and also by the effects of an altered Ca2+ concentration of the medium. The developmental changes in cGMP fit with this model. Thus, the Ga, peak in cGMP is presumably the result of an increased Ca2+ influx induced by CAMP. The Ga, peak, on the other hand, may be caused by an increased acetylcholine formation (cf. Buznikov et al., 1968). The effects of muscarinic agents on the cGMP contents, however, are not consistent with this model. As has been suggested in the Results section, however, the effects of added muscarines on cGMP may be explained by the occurrence of two fractions
Pharmacological control of cilia of guanylcyclase, a minor one bound to the cell surface and controlling the Ca*+ influx, a major one of endocellular location and indirectly activated by the Ca*+ influx into the cell. The suggested interaction between the two cyclase systems, the feedback inhibition of the adenylcyclase, and the suggested interplay between a cell membrane linked and an endocellular pool of guanylcyclase, all presumably related to the cellular content of ionic Ca*+, an ion that presumably also plays a direct role in cellular excitation, may still be considered a working hypothesis.
Acknowledgements-This paper is dedicated to my teacher, Professor Tryggve Gustafson, to whom I would like to express my cordial thanks for encouragement, stimulating discussions and valuable criticism during the experimental studies and the preparation of the manuscript. Many thanks are directed to Professor Jarl-Ove Stromberg and the staff of Kristineberg’s Marine Biological Station for working facilities and supply of material. The skilful assistance by Mrs Anna-Lena Kullgren and the technical advice by Miss Marie-Louise Tjornhammar is gratefully acknowledged. I am also grateful to Mrs Renate Ahlfont for typing the manuscript. This project has been supported by a grant from the Swedish Natural Science Research Council to Tryggve Gustafson. REFERENCES Brown
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