Camp. Biockm. Physid. Vol. 89C, No. 2, pp. 185-I 89, 1988 0306-4492/88 $3.00 + 0.00

Printed in Great Britain 0 1988 Pergamon Press pie

RECOVERY OF ACETYLCHOLINESTERASE ACTIVITY AFTER IRREVERSIBLE INHIBITION

BY ORGANOPHOSPHOROUS COMPOUNDS IN EMBRYONIC DEVELOPMENT

T. M. TURPAEV and M. N. SEMENOVA

N .K. Koltzov Institute of Developmental Biology, USSR Academy of Sciences, Moscow, USSR

(Received 3 Februury 1987)

Al&ret-I. Recovery of a~tylcholinesterase (AChE) activity was studied using the embryos of sea urchins Strongylocentrotus Intermedius and S. n&us, embryos of axolotl Ambystoma mexicanurn and in the chick embryo muscle culture treated by “irreversible” organophosphorous inhibitors (OPI).

2. AChE activity was assayed by a modified Ellman’s procedure. 3. It follows from the data obtained that, unlike the plutei of sea urchins and the monolayer culture

of chick embryo muscle cells, the embryos of axolotl show a compensatory increase in AChE biosynthesis after inhibition by OPI.

4. This mechanism is assumed to be related to the presence of a well developed neuromuscular system in the A. mexicanurn embryos.

5. It is possible that acetylcholine accumulated as a result of partial AChE inhibition is responsible for the compensatory increase in AChE biosynthesis.

It has been shown that the components of the choli- nergic system (acetylcholine, acetylcholinesterase, acetylcholine receptors) are found at the early “pre- nervous” stages of embryonic development or even in the unfertilized egg (Buznikov, 1980; Fluck, 1982; Semenova and Turpaev, 1985). In this connection of special interest are studies on a nonsynaptic role of the cholinergic system during embryogenesis. It has been suggested that the cholinergic system is directly involved in the interactions of embryonic cells and responsible for morphogenesis and motor activity of the embryo (McMahon, 1974; Drews, 1975; Vanit- tanakom and Drews, 1985). An important part in cholinergic system function is played by acetylcholin- esterase (AChE, EC 3.1.1.7.), an enzyme which hydrolyses acetylcholine (ACh). It is known that intensive synthesis of AChE begins at a certain stage of embryonic development and the activity of this enzyme markedly increases. The process appears to be triggered by a specific gene after the cell passes a certain number of DNA replication cycles (Satoh and Ikegami, 1981; Mita-Miyazawa ef af., 1985). Further increase in AChE activity is usually associated with the development of the motor system and di~erentiation of embryonic neuro-muscular com- ponents. The AChE activity is closely correlated with the motility of single embryonic cells and the whole embryo (Sawyer, 1943; Gustafson and Toneby, 1971; Drews, 1975; Minganti et al., 1979; Semenova and Turpaev, 1983).

In order to elucidate one of the regulatory mech- anisms of AChE activity we decided to use organo- phosphorous compounds which irreversibly inhibit this enzyme. Using cultured chick embryo muscle cells Cisson and Wilson have shown that the rate of AChE synthesis after the inhibition by paraoxon is

directly proportional to the degree of the enzyme inhibition. They supposed that there exists a feedback mechanism between AChE activity and the rate of its biosynthesis (Cisson and Wilson, 1977). To test the hypothesis we have undertaken experiments to char- acterize the recovery of AChE activity in the em- bryogenesis of various animal species after the in- hibition by organophosphorous inhibitors (OPI). We used early sea urchin embryos, where the presence of the cholinergic nervous system has not been estab- lished (Ivanova-Kazas, 1978); embryos of Amby- stoma having a well developed neuromuscular system (Boell and Shen, 1950; Hughes, 1968); and mono- layers of chick embryo muscle cells which provide a means of evaluating changes of AChE activity in early myoblasts differentiation. The experimental ma- terial is known to contain a specific cholinesterase, or AChE (Wilson et al., 1973; Semenova and Turpaev, 1983). The following organophosphorous com- pounds were used as irreversible inhibitors of AChE: Gd-7 (Volkova et al., 1961), GT-106 (Brestkin et al., 1968), V-81 (Vikhreva et al., 1980), V-156 (God- ovikov et al., 1984). Hydrophobic properties of these inhibitors allow them to enter cells or the whole embryos.

MATERIALS AND METHODS

Anim& and culture techniques

Developing sea urchin Strongylocentrotus intermedius and S. nudus embryos were obtained by the described procedure (Buznikov and Podmarev, 1975) which was also used to define embryonic stages. AChE activity was assayed at the hatching (middle blastula stage) to the beginning of active feeding (middle pluteus 2). Before measurements the em- bryos were centrifuged for 10min at 0°C and 3OOOrpm to concentrate the suspension.

185 f a P s9;2c-E

186 T, M. TURPAEV and M. N. SEMENOVA

The eggs of Ambystoma mexicanum were incubated at room temperature (18-21’C) in tap water. Stages of em- bryonic development were determined according to Bordzilovskaya and ~e~a~(l975~. At stage 35-?6 when the embryos began to show a response to pricking by a needle, they were mechanically freed from the enveIopes, and the incubation continued with daily changes of water. AChE activity was assayed from the beginning of the motor response to the pricking of a needle (stage 35-36) to the stage of mouth opening (stage 43). It should be noted that the intact and treated with inhibitor Ambystuma embryos were kept in the same aquaria and in roughly the same rmmbers to insure against the effect of the aquarium size and the population density on the size of embryos.

The monolayer culture of chick embryo muscle ceils was obtained from the hind limb of 1 l-day-old chick embryos (Lyzlova et al., 1971). During trypsinization we used phos- phate buffer according to Andzhaparidze et al. (1962). For &lturing, IOml of Cell suspensiOn in a concentration of 2-6 x lO-$ cellsiml was niaced in a IO cm Petri dish, and the culture was in&bated in medium 199 supplemented with 7% bovine serum, 3% chick embryo extract made from IO-day-old embryos, penicillin G (200 units/ml) and strepto- mycin (100 units/ml). The culture medium was first changed a day after plating and then every 2 days. The cells were cultured for 4-5 days, and by the end of this period spontaneous contractions of myotlbrills and the detachment of monolayer from the glass were usually observed.

AC!& assay

AChE activity was measured by a modified Ellman’s procedure (Gorun et al., 1978) in homogenates prepared with the use of a e;lass homogenizer with a Teflon pestle in a 0.02 M phosphate buffer, pH 7.6 (the eggs of sea urchins were ~ornogen~~ in the filtered seawater). WheB necessary, the homogenates were kept frozen for 2-8 days which had no effect on AChE activity. In such cases homogenization was repeated after thawing. The sample contained 20 ~1 of homogenate: 10-35 thousand embryos per ml for the sea urchins; 2 embryos per 0.8 ml for axolotl and 0.1-0.3 ml phosphate buffer/Petri dish for the culture of chick embryo muscle cells. Concentration of the substrate (acetyl- tbi~holine~ was 10-~ M for the Ambysf~rn~ embryos and mu&e tissue cufture and 5 x 10m3 M for the sea urchin embryos. Enzymatic reaction was run at 25*C, pH 7.6 for 30 min. AChE activity was expressed in pMN/embryo/hr or in FM/Petri dish/br. Since the rate of embryonic devel- opment and of the myablast culture growth varied between experiments, to illustrate the changes in AChE activity we have chosen the curves obtained for a single sample of embryos and typical for the subject under study.

OPI treatment The embryos of sea urchins at the stage of early pluteus

1-2 were treated with V-81, CT-106 or Gd-7 (2 x 10-‘-l x 1O-3 M) for 1.5-3 hr, and then washed with seawater on the nylon gauze with the pore diameter of 90 p. Arn~~~f~rnu embryos at stage 38-39 were treated with Gd-7 (2 x lW6-1 x 1W4 M) far 15-30 min and then washed care- fully. The treatment of chick myobiasts with V-156 was usually performed 2 days after culturing when proliferation was completed and myasyncytium formed. The recovery of AChE activity in the muscle cells was observed after 15 min treatment with V-156 (2.5 x lo-C5 x 101~ M), after which the cells were washed three times with medium 199 and incubate further in the culture rn~~~. The efficiency of washing was controlled in the following way. After washing OPI-treated embryos or myoblast, culture were placed for 20min in the water bath (8O”C), then homogenate was prepared and added to the homogenate of intact embryos or culture. In none of the cases was AChE activity of intact homogenate decreased implying that there was no active inhibitor. To exclude a po~ibiiit~ of a spontaneous reac-

tivation of the phosphorylated enzyme, the homogenates from OPI-treated embryos or muscle tissue culture were kept at 4°C for 7-8 days with daily measurements of AChE.

The chemicals used were a~tyit~~oeholine iodide (CHEMAPOL, Czechoslovakia); 5,s’.ditltyo-bis-2-nitro- benzoic acid (SERVA, BRD); Gd-7 [O-ethyl-S@-ethyl- mercaptoethylmethyl)phosphothyonate], GT-106 [0,0-di- ethyl-S(B-methylcyclohecksylaminoethyl)phosphothyoate], V-81 [~,~-djethyl-~(o~-mercaptoethylb~t-2-ynyl)phos- pho~~oate], V-156 EU,o-die~yi-s(om-acetoxybut-2-yn~l~” phospho~hyoate] (OPI were s~~th~~~~ in the Institute of Organoelemental Compounds, USSR Academy of Sciences).

RESULTS

In the plutei treated with Gd-7, GT-106, V-81 the AChE activity decreased to 7040% of the original ane depending on the concentration, the duration of treatment and the inhibitor. As early as 2.5-3 hr afier treatment AChE activity increased roughly in parallel to the increase in the e~me level in the intact embryos, and the normal level of activity was not attained until at least the middle pluteus 2 stage (Fig. 1). The experiments with two species of the sea urchins with all three inhibitors gave similar results. It should be noted that the decrease in AChE activity in the OPI-treated plutei produces no apparent developments abno~al~ties or motor activity dis- turbances up to the stage of the active feeding.

Recovery of ACThE activity in the monolayer culture of chick embryo muscle cells after the treatment with V-1.56

A low activity of AChE is found in the chick embryo myoblasts before culturing. Approximately f day after culturing the AChE activity begins to increase which coincides with the beginning of fusion of myoblasts to myotubes, the process is seen well under microscope; then, as the muscle tissue differentiation proceeds, the activity of AChE in- creases up to a certain level at which it remains unchanged until the monolayer comes off the glass

&gef hr

Fig. 1. Effect of V-81 on AChE of S. intwmedius plutei. (1) AChE activity in intact embryos. (2) The same in the embryos treated with V-81 (2.5 x lo-’ M) for 180 min. The arrow corresponds to the end of tmatment. Confidence

intervals are given for the 5% s~ifi~n~ level.

Embryonic AChE recovery after OPI inhibition 187

Fig. 2. Recovery of AChE activity in the culture of chick embryo muscle cells after 15 min treatment with V-156, 2.5 x 10d6M (2) and 1 x lo-‘M (3). (1) Change in AChE activity in the intact culture. Confidence intervals are given for the 5% significance level. The arrow shows the moment

of treatment.

(curve 1, Fig. 2). After the treatment with V-156 AChE activity first decreases and then begins to increase up to a certain maximal level, not attaining, however, the level of AChE in the intact culture (curves 2 and 3, Fig. 2). Then the activity of AChE decreases again. In the chick embryo muscle cells culture the increase in AChE activity after the treat- ment with V-156 proceeds more slowly than in the intact culture, and the maximal recovery of the enzyme activity, observed 2 days after treatment, corresponds in time with the moment when AChE in the control culture reaches its constant level. When the muscle culture was treated with various concen- trations of V-156 it was found that the lower the inhibitor concentration, the lower the degree of AChE inhibition and the quicker and more complete the recovery of its activity (Fig. 2). Though the recovery of AChE activity is incomplete, there are no differences between the treated and untreated cultures either in the morphological characters and protein content, or in the time of appearance of the sponta- neous contractions of muscle fibers.

Recovery of AChE activity in the Gd-7-treated Ambystoma embryos

In the ~~~~sto~~ embryos treated with Gd-7 at

t 2 ii3

5 I *2

% .z

3 I

9

4"

0 , days

f371m3) 139) MO) (421 (4343) Stage

Fig. 3. Recovery of AChE activity in the embryos of Ambystoma mexicanurn treated with Cd-7 in concentration of 5 x IO-” M (2) and 1 x 10v5 M (3) at stage 39 during 30 min (1). Change in AChE activity in intact embryos. The arrow shows the moment of treatment. The asterisk marks the AChE activity values from curves 2 and 3, with significant differences between them. Each point on the

graph is a mean of 36 measurements.

stage 38-39 the AChE activity decreases by !SlOO%, depending on the inhibitor concentration, the duration of treatment and individual sensitivity of each batch of embryos to Gd-7. The decrease is accompanied by a disturbance of motor activity up to the loss of the embryo response to mechanical stimu- lation. However, such a decrease of the AChE activ- ity does not affect embryonic development until the stage of mouth opening (stage 43). It was only occasionally that we observed a slightly decreased pi~entation in the treated embryos at stage 42-43. Usually during the first day after treatment there was practically no change in the AChE activity. Two days after the treatment the AChE activity increased noticeably reaching the control level in 5-7 days (Fig. 3). The increase in the AChE activity is paralleled by the recovery of the ability of embryos to swim. In all experiments the rate of the rise of AChE activity in the Gd-%treated embryos exceeded that in the intact ones and was directly proportional to the level of the original AChE inhibition, as seen in the experiments using various concentrations of Cd-7.

DISCUSSION

It is now generally assumed that the recovery of the AChE activity in the animals or tissue cultures treated with OPI is due to its biosynthesis (Harris et al., 1971; Cisson and Wilson, 1977, 1981). In our experiments we found no increase in homogenates incubated at 4°C from OPI-treated ~~~y~t~~a and sea urchin embryos and from the culture of chick embryo muscle cells. This implies that the phos- phorylated AChE is not spontaneously reactivated and the recovery of AChE activity occurs by its synthesis de nova. The absence of spontaneous reac- tivation of AChE is further supported, though indi- rectly, by the observation that during the first 24 hr after the Gd-7 treatment of Ambystoma embryos the enzyme activity increases only slightly if at all (curves 2 and 3, Fig. 3). The results obtained with the sea urchin plutei and the culture of chick myoblasts (Figs 1 and 2) show that the rate of recovery of AChE activity after treatment with OPI is equal to or lower than the rate of increase of AChE activity in control. It can be said, therefore, that in the plutei of S. intermedius and S. nwdus and in the monolayer cul- ture of chick myoblasts no compensatory increase of AChE biosynthesis takes place after its inhibition by irreversible inhibitors. Besides, we have found the following trend when treating the culture of chick myoblasts with various concentrations of V-156: the higher the level of AChE inhibition, the slower the process of the recovery of its activity; and vice versa, the lower the percentage of inhibition, the quicker the recovery of AChE activity reaching a higher level (curves 2 and 3, Fig. 2). This is further evidence for the absence of feedback between the activity of AChE and the rate of its biosynthesis in the monolayer culture of chick myoblasts. It is possible that the absence of feedback is characteristic just of the early stages of muscle tissue differentiation, whereas such feedback is involved in the control of AChE activity in the multilayer culture at later stages (Cisson and Wilson, 1977).

188 T. M. TURPAEV and M. N. Smmov~

It should be noted that in a more differentiated of ACh, since it takes several days for the enzyme multilayer culture of chick embryo muscle cells the activity to reach the control level, and the recovery activity of AChE after the treatment with OPI recov- ers much quicker than in the monolayer culture

does not begin until 2 days after the inhibition (Fig.

(Walker and Wilson, 1976; Cisson and Wilson, 198I), 3). If we assume that ACh is involved in the compen-

sometimes reaching the control level in several hours satory increase of the AChE biosynthesis after the

(Cisson and Wilson, 1977; Golder et al., 1978). inhibition by OPI, it becomes clear why there is no feedback between the activity of AChE and the rate

Of special interest is the decrease in AChE activity of its biosynthesis in the plutei of sea urchins where in the culure treated with V-156, 3 days after the no cholinergic nervous system was found (Ivanova- treatment (Fig. 2). A similar observation was made Kazas, 1978) and, therefore, ACh cannot play the after the treatment of multilayer muscle cells culture role which it plays in the Ambystoma embryos that with diisopropylphosphofluoridate. Thus, 4-6 hr have a well developed neuromuscular system (Boell after the treatment with diisopropylphosphofluor- and Sheen, 1950; Hughes, 1968). idate the recovery of the AChE activity is maximal, after which the activity slightly decreases due to the Acknowledgements-The authors thank Professor A. V. enzyme secretion into culture medium (Walker and Zhirmunsky and Dr V. L. Kasyanov for providing the

Wilson, 1976). It is possible that the same occurs in opportunity to work on the biological station “Vostok”. We

the monolayer chick embryo muscle cells culture after are grateful to Dr N. N. Godovikov for kindly supplying us

the treatment with V-156. In our experiments AChE with organophosphorous inhibitors of acetylcholinesterase,

activity recovers slower than in the multilayer culture Dr Eh. I. Bueverova for the assistance in the work with

and the maximum of the recovery occurs roughly 3 tissue culture, Dr I. N. Sokolov for the cons~~tion on the culture of chick mvoblasts and Dr L. A. Gudkov. the head

days after the treatment, instead of 4-6 hr in the of the Aquarium of the Institute of Developmental Biology, multilayer culture. Obviously, further studies are USSR Academy of Sciences, for providing experimental necessary to find out the cause of the decreased material.

AChE activity in the treated culture; we think now that this phenomenon is unrelated to the toxic effect of V-l 56. It may be assumed that this decrease is the REVERENDS

result of the intensive secretion of the enzyme into the culture medium that begins after the maximum of the

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