recovery of activity and molecular forms of cholinesterase in different animal species following...

Comp. Biochem. Physiol, Vol. 71C, pp, 27 to 35, 1982 0306-4492/82/010027-09503.00/0 Printed in Great Britain. All rights reserved Copyright © 1982 Pergamon Press Ltd

RECOVERY OF ACTIVITY A N D MOLECULAR FORMS OF CHOLINESTERASE IN D I F F E R E N T ANIMAL SPECIES F O L L O W I N G IRREVERSIBLE

CHOLINESTERASE INHIBITION

A. MIKALSEN, R. A. ANDERSEN,* J. A. B. BARSTAD and G. LILLEHEIL Department of Toxicology, National Institute of Public Health, Postuttak, Oslo 1, Norway

(Received 29 April 1981)

Abstract--1. Diisopropoxyphosphorylfluoride and soman caused rapid decline of the cholinesterase (ChE) activity levels in earthworm, chicken, guinea pig, mouse and rat tissues.

2. The onset of recovery of ChE activity was well established within 5 days in all species except for the earthworm whose whole body ChE activity level remained low for at least 15 days.

3. Plasma ChE of the rat recovered to the normal activity level already in 6 days. After that, however, the activity level still continued to increase. This overshoot effect was much more pronounced in females than in males.

4. In females the activity level reached 50~o above normal values after about 7 days. The overshoot of plasma ChE in the female rat still persisted 15 days after inhibition.

5. The overshoot of ChE activity was also observed in mouse and rat liver after ChE inhibition. In the chicken ChE overshoot was not found.

6. The recovery of erythrocyte and brain ChE activity of the rat was observed to be slower than that of plasma and liver. Likewise the recovery of brain ChE activity of the mouse seemed to be slower than that of liver. Similar results were obtained for the chicken. Generally soluble ChE seem to recover faster than membrane bound.

7. In the case of erythrocyte and brain ChE the results indicated a similar ChE turnover as generally observed for other proteins in brain tissue.

8. In brain and retina of chicken as well as in rat brain and in guinea pig iris two distinct forms of ChE were found with marked different molecular weights.

9. In the chicken the activity of the form with a low molecular weight was found to predominate during the early recovery stages following ChE inhibition. At later stages the activity of a heavy weight form dominated.

10. In rat brain and retina as well as in guinea pig iris the activity of the form with the higher molecular weight was always predominating.

11. The results may, however, be interpreted such that the synthesis of the form of ChE with the lower molecular weight proceeds the appearance of the higher molecular weight form.

12. Administration of L-anserine nitrate, L-homocarnosine sulphate, L-carnosine and thyrotropin releasing hormone to the rats as well as primobolan to the mouse did not affect the recovery of activity and molecular forms of ChE following irreversible ChE inhibition. In the rats hypophysectomy was not found to affect the recovery either.

I N T R O D U C T I O N

Molecular forms or isozyme composi t ion of cholin- es teraset (ChE) have been studied by several au thors ; Massouli6 & Rieger (1969), Crone (1971), Hollunger & Niklasson (1973), Wentho ld et al. (1974), Vijayan & Brownson (1974), Dudai & Silman (1974), Anglister & Silman (1978), Andersen & Mikalsen (1978a, b, 1979). Where molecular forms of particle bound ChE have been studied, solubilization most commonly has been accomplished by the use of the unionic detergent Tri ton X-100.

Previously activity and molecular forms of ChE in animal developmental stages have been studied among others by Maynard (1968), Rieger & Vigny (1976), Marchand et al. (1977) and Henderson (1977). Recovery of ChE after inhibi t ion in whole animals and tissue cultures have been studied by Clouet &

* To whom reprint requests should be addressed. t See Chemicals for abbreviations.

27

Waelsch (1961), Austin & James (1970), Harris et al. (1971, 1974), Wilson & Walker (1974), Yaksh et al. (1975) and Rieger et al. (1976). To discriminate between recovery of ChE activity caused by reacti- vat ion of inhibited enzyme and new synthesized enzyme it is necessary to choose a fast aging inhibitor. The well known nerve gas soman as well as D F P have been used in our experiments (Heilbronn- Wiks t rom (1965), see also Materials and Methods).

In the present work recovery of activity and molecular forms of ChE in the ear thworm, chicken, guinea pig and rat following inhibi t ion by soman has been studied. Some corresponding experiments on recovery of ChE activity have been performed with D F P in the mouse. Particle bound ChE has been solubilized by Tr i ton X-100 and molecular forms separated by polyacrylamide gel electrophoresis and sucrose gradient centrifugation. These experiments have been performed in order to gain further infor- mat ion about ChE turnover in different animal species and to throw light on the mechanisms by which

28 A. MIKALSEN et al.

ChE is synthesized and developed into functional forms in the body. Some preliminary experiments concerning a possible regulation of these processes have also been performed.

Previously it has been demonst ra ted that the ChE activity in plasma recovered to activity levels considerably higher than normal following irreversible ChE inhibition, Laake & Smith (1975), S~li et al. (1977). This phenomenon has been investigated further in the present work.

MATERIALS AND METHODS

Solubilization

In the present study the non-ionic detergent Triton X-100 has been used to solubilize ChE from cerebral cortices and retinas from chicken and rat and from guinea pig iris musculature. This detergent is believed to be advan- tageous for solubilization of membrane bound enzymes and in studies of isozyme composition, because it generally does not seem to induce conformational changes in proteins leading to loss of biological properties (Helenius & Simons, 1975). Triton X-100 also seems to be very effec- tive in solubilizing particle bound ChE from different animal species (Andersen, 1979).

Inhibition and aging Because the return of ChE activity, after inhibition by

soman and DFP, is thought to be due to de novo synthesis of ChE, the reappearance of enzyme activity should give an approximation of the turnover rate of ChE in the tissues. This is based mainly on two assumptions: (1) the inhibition by soman and DFP is irreversible and (2) there is no free inhibitor present to combine with the newly formed enzyme at the dose level used. After inhibition by soman the ChE-inhibitor complex ages, i.e. it loses the pinacolyl group (in DFP the isopropyl group) to form a product that becomes refractory to reactivation (Heilbronn-Wickstr6m, 1965; Berry & Davis, 1966; Harris et al., 1971). The aging of the ChE-soman complex seems to be exceptionally fast. The t½ value of aging of monkey brain ChE is 1 min (Fil- bert et al., 1972), while it is 2,4 min in bovine erythrocyte ChE (Fleisher & Harris, 1965). In addition Fleisher & Harris showed that there was no loss of the residual meth- ylphosphonate from the aged enzyme in vivo up to 72 hr. For DFP the aging t½ values have previously been found by Andersen et al. (1972) to be 2,3 and 2,6 hr for mouse and rat brain ChE, respectively. Results gathered by Aldridge & Reiner (1972) indicate that the rate of aging do not differ considerably between membranebound and soluble plasma ChE for different inhibitors. The absence of free soman and DFP in the tissues was verified by incubation of aliquots of tissues from poisoned animals with aliquots taken from tissues of normal animals. The enzyme in tissues from normal animals was not inhibited under these conditions. Soman and DFP also seem to be without effect on protein synthesis (Harris et al. (1974)).

Chemicals The substrates for ChE, namely acetylthiocholineiodide

(AThChI), propionylthiocholineiodide (PrThChI) and butyrylthiocholinechloride (BuThChC1) as well as 5,5'-dith- iobis-(2-nitrobenzoic acid) (DTNB), the unionic detergent Triton X-100, atropine sulphate, the marker enzyme catalase and the compounds L-homocarnosine sulphate, L-anserine nitrate, L-carnosine, L-pyroglutamyl-L-histidyl- L-proline amide (thyrotropin releasing hormone) were all from Sigma Chem Co. The inhibitors soman (pinacolyloxy-

* Kindly performed by J. Blanch and S. Oksne, Division for Toxicology, Norwegian Defence Research Establish- ment, Kjeller, Norway.

methyl phosphorylfluoride) and DFP (diisopropoxyphosphorylfluoride) were prepared in minute amounts. Their purity was controlled by nuclear magnetic resonance spec- tra to be more than 98~o*. Sucrose of high grade purity was obtained from Merck. The reagents used in polyacrylamide gel electrophoresis, namely acrylamide, N,N'-methy- lene-bisacrylamide, N,N,N',N'-tetramethylethylenediamine were from Eastman Kodak Co., ammonium peroxodisul- phate from Baker Chem., NV, glycine from BDH Chem. Ltd. and tris(hydroxymethyl)-aminomethan from Merck. The anabolic steroid primobolan (primobolan-depot) was obtained from Schering Corp.

Procedures and enzyme preparations Specimens of the earthworm species, Eisenia foetida, of

body length 5-8 cm, were purchased from the bait dealer Bunes Spesialkulturer, Verdal, Norway. The worms were rinsed in tap water, cleansed by leaving them on wet filter paper for 4 days, then rinsed, weighed, minced and hom- ogenized in 0.05 M phosphate buffer (10~o w/v), pH 7.4 at 1400 rpm and 2-5°C with a glass-Teflon homogenizer. The crude homogenate was used for determination of ChE activity (see Andersen et al. (1978a) for a characterization of ChE from Eisenia foetida).

Cerebral cortices and retinas from chicken (Gallus domesticus) (6 weeks old) and rat (Rattus norwegicus ) as well as iris musculature from guinea pig (Carla porcellus) were dissected out and crude homogenates prepared as above. For the mouse (Mus musculus) whole brain was used. The homogenates were then mixed with 0.25~o (v/v) Triton X-100 for 45 min at 5°C to let the ChE solubiliz~. The supernatant collected after centrifugation at 100,000 g for 1 hr constituted the solubilized ChE. Blood from chicken and rat was collected and centrifuged to separate erythrocytes and plasma. The erythrocytes were washed in physiological saline and plasma centrifuged at 100,000g for 1 hr before use. Crude homogenates from mouse and rat liver were prepared as above.

Brain, retina, iris and liver tissue as well as crude homogenates and freshly prepared solubilzed ChE preparations could be stored frozen at -20°C for at least 1 week without change in enzyme activity and molecular form patterns. This was also observed by Marchand et al. (1977) for chicken brain tissue.

The ChE of the earthworm was inhibited by immersing specimens into a solution of 1.25.10- 5 M soman in water for ½ min. The worms were then rapidly cleaned by rinsing them in tap water. Chicken and rat ChE was inhibited by s.c. injection of the LD~0 dose of soman and DFP in physiological saline. For soman these doses were found to be 0.01 mg/kg for the chicken and 0.1 mg/kg for the rat, while for DFP in the mouse it was found to be 4 mg/kg. To keep most of the treated animals alive atropine sulphate (5 mg/kg) was given s.c. 30 min prior to inhibitor administration. The selective inhibition of the ChE of the guinea pig eye was performed by corneal application of 2/A soman in physiological saline (1 mg/ml). Soman and DFP were otherwise kept in isopropylalcohol (1 mg/ml) as stock solutions.

L-anserine nitrate, L-homocarnosine sulphate, L-carnosine and thyrotropin releasing hormone were administered to the rats coincidentally with the inhibitor and then subse- quently each day during the ChE recovery time. The compounds were given s.c. in physiological saline in doses of 10 mg/kg except for the dose of thyrotropin releasing hormone which was 5 mg/kg. Primobolan was given in a single dose of 10 mg/kg to the mice coincidentally with the inhibitor. Hypophysectomised rats were obtained from Charles River Inc., France.

ChE activity measurements

The method of Ellman et al. (1961) was generally used to

Recovery of ChE after soman inhibition

Table 1. Normal ChE activity levels in different species

Activity ChE mmol AThCh/ml/min* ttmol AThCh/mg prot./min

Earthworm whole body 0.72 0.063 Chicken plasma 0.61 0.015

erythrocytes 0.28 0.0008 cortex 1.04 0.094 retina 1.44 0.28 retina]. 1.40 0.27

Rat plasma 0.32 0.0048 erythrocytes 1.06 0.0035 cortex 1.50 0.13 cortex]. 1.70 0.15 retina 0.80 0.10 retinal" 0.98 0.12

Mouse brain 1.34 0.10 liver 0.16 0.0091

Guinea pig iris 0.16 0.012 iris] 0.14 0.0098

* For brain, retina, liver and iris the values represent 10~o (w/v) raw homogenate unless specified. For erythrocytes the activities represent a 10~o (v/v) suspension, prepared by centrifugation at 750 g for 10 min (see also Materials and Methods).

]. Supernatant after ChE extraction by 0.25~o (v/v) Triton X-100.

29

determine ChE activity. For blood a modification of this method was used (Andersen et al., 1978b).

Density gradient centrifugation The procedure described by Martin & Ames (1961), was

used. Sedimentation in sucrose gradients (5-20~o w/v, total vol 4,8 ml) was performed by centrifugation at 29,000 rpm for 15 hr at 4°C in a Beckman ultracentrifuge, model L5-65 with the SW-50,1 rotor (see also Gritfith (1975) for details and experimental procedures). Fractions were collected from the bottom of each centrifuge tube. For estimation of sedimentation coefficients (S values) and approximate molecular weights catalase (11,4 S) was used as marker. Cata- lase activity was measured as described by Martin & Ames (1961) and ChE activity of each fraction as described by Ellman et al. (1961).

Except for runs with chicken and rat sera the sucrose gradients were incorporated with 0.25~o (v/v) Triton X-100. The detergent in this concentration, however, was not found to influence the molecular form pattern of the sera.

Polyacrylamide #el electrophoresis The method described by Weber & Osborn (1975) was

used. Electrophoresis was performed in glass tubes with a length of 6.5 cm and an i.d. of 0.5 cm. The gels were not equilibrated with detergent. The staining method of Kar- novsky & Roots (1964) was used to visualize the ChE bands after electrophoresis.

Protein measurements The measurements were performed according to the

method of Kjeldahl using the Biichi 425 digestor and 315 distillation unit. Selenium mixture (Wieninger) was used as catalysator.

The numerical values given in this work are based on at least 4 determinations. The level of significance (P) was 0.05.

RESULTS

In Table 1 the normal ChE activity levels deter- mined in different tissues are listed. As expected the highest values were found in nervous tissue.

Treatment of the animals with the irreversible inhibitors soman and D F P caused rapid decline of ChE activity in all tissues investigated. The inhibition occurred within the first 2 hr after treatment and no appreciable change from this level was observed during the following 10 hr. Thus the enzyme activity at 12 hr (clay 1) after treatment could be taken to represent the maximum degree of inhibition. Free soman or D F P did not remain in the animal tissues 2 hr after treatment. The ChE activity levels in different tissues 12 hr after treatment with the LDso dose of the inhibitor are given in Table 2. The ChE in the liver of the rat and the mouse was not inhibited to the same extent as the ChE in other tissues of the same ani-

Table 2. ChE activity levels in different species in ~o of normal 12 hr after irreversible ChE inhibition by the LDso dose

Whole body Cortex Retina Liver Plasma Erythrocytes Whole blood

Earthworm Chicken Rat

Q + S

Mouse*

16 28 32 - - 37 5 - -

29 62 61 17 16

43 66 63 12 14 - - 64 - - - - 23

* For mouse DFP was used as ChE inhibitor, for the other animals soman.


7,

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60

20

100

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140

100

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Fig. 1. The time--course of ChE recovery in different species following irreversible ChE by the LDso dose. (a) earthworm whole body, (b) chicken serum, (c) rat cortex, (d) rat erythrocytes, (e) rat liver, (O rat serum, (g) mouse brain and liver. Males--O---, females - - O - - - , mouse b r a i n - - O - - , mouse liver + . For mouse DFP was used as ChE inhibitor, for the other animals soman. The results are

presented within 95Yo confidence limits.

reals. The new synthesis of ChE following inhibition seemed to be well established after 5 days except in the case of the earthworm whose whole body ChE activity remained low at least for 15 days~ Figure 1, a -g show the time course of ChE recovery in different animals and tissues following ChE inhibition by soman and DFP. In Table 3 the half time values of recovery are specified. The increase towards the normal activity levels of erythrocyte and brain tissue ChE of the rat was observed to be slower than that of plasma and liver ChE. Likewise the recovery of brain ChE activity of the mouse seemed to be slower than that of liver. Similar results were obtained for the chicken.

Plasma ChE of the rat reached the normal activity level already in 5 days. After that, however, the plasma ChE activity level still continued to increase. This overshoot effect was found to be much more pronounced in females than in males. In females the activity reached levels 50~o above normal values while in the males only a moderate, transient overshoot could be demonstrated. Plasma ChE activity levels of the female rat still remained high 15 days after soman inhibition. Such an overshoot effect after ChE inhibition by soman was not shown in the chicken. The

Recovery of ChE after soman inhibition 31

Table 3. The time to reach 50~o recovery (t½) of the ChE activity in different species following irreversible ChE

inhibition

ChE Days

Chicken plasma 3.7 erythrocytes 11.6 cortex 8.0 retina 4.5 plasma ~ 4.3

Rat 3 ̀3.4 12.6

erythrocytes 3' 10.0

cortex ~ 12.3 3` 8.0

retina 8.8

liver ~ 2.0 3 ̀2.5

Mouse brain 11.0 liver 2.1

Guinea pig iris 1.8

For mouse DFP was used as ChE inhibitor, for the other animals soman.

effect seemed, however, to be present in the mouse following D F P inhibition.

Nei ther the total amount of b lood nor the relative amount of plasma compared to erythrocytes in

0,3

= I 0'2

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Fig. 2. Velocity sedimentation in linear sucrose gradients (5-20% w/v) of ChE solubilized by 0.25% (v/v) Triton X-100 (unless specified) from various species at different time intervals following inhibition by the LDs0 dose of soman. (a) chicken cortex, normal - - l - - , day 1 - - l - - , day 9 ---O--, day 22 - - O - - . (b) chicken retina, normal ---tl---, day 1 - - I - - , day 10 ---4D---, day 23 - - O - - . *(c) chicken serum, normal - - 1 - - , day 4 - - l - - , day 5 ---O--, day 7 - - O - - . (d) rat cortex, normal - - l - - , day l - - l - - , day 14, - -O-- , day 27 - -<3- - . (e) rat retina, normal - - I - - - , day 1 - - I - - , day 14 - -O-- , day 27 - - O - - . *(f) rat serum, normal - - I - - , day 1 - -O-- , day 8 - - O - - . (g) guinea pig iris, normal - - I - - , hr l - - i - - ,

hr 24---0-- , hr 4 8 - - O - - . * Not detergent treated. Sedimentation was performed in gradients incorporated

with 0.25% (v/v) Triton X-100. AThCh was used as substrate in the ChE activity determinations. The abscissa indicates volume of gradient collected from bottom of the

centrifuge tube.


Table 4. Sedimentation constants (S) and corresponding mol. wt of molecular forms of ChE in different species separated by sucrose gradient centrifugation after solubilization of the ChE with 0.25% (v/v)

Triton X-100

ChE S (mol. wt)

Earthworm Chicken

Rat

Guinea pig

6.5 + 0.23 (5) (108,000 + 6,000) plasma 11.1 + 0.81 (5) (243,000 + 27,000) cortex 11.6 + 0.43 (4) (260,000 + 15,000) 7.8 + 0.21 (5) retina 11.7 + 0.39 (4) (263,000 + 13,000) 7.7 + 0.25 (4) plasma 10.9 + 0.30 (5) (237,000 + 10,000) cortex 10.8 + 0.29(5) (234,000 + 9000) 4.8 + 0.19 (5) retina 11.0 _+ 0.37 (4) (240,000 + 12,000) iris 10.8 _+ 0.25 (5) (234,000 + 8000) 4.8 _+ 0.18 (5)

(143,000 + 6000) (141,000 _+ 7000)

(69,000 _+ 4000)

(69,000 + 4000)

The results are presented within 95% confidence limits.

chicken and rat were found to change during the experiments. Animal weight changes were not observed either.

Plasma ChE of chicken and rat could not be separated into different isoenzymes by sucrose gradient centrifugation. It was not possible to demonstrate such a separation at any stage of ChE recovery following inhibition by soman either (Fig. 2f). The molecular weights of plasma ChE in the chicken and rat are given in Table 4.

In chicken brain and retina, however, the ChE activity could be separated by sucrose gradient centrifugation into one large activity peak and a small shoulder thereby indicating that the ChE solubilized by Triton X-100 from the chicken brain and retina consists of at least two forms (Fig. 2a,b). Correspond- ing forms of brain and retina did not differ with respect to S values. See Table 4 for S values and molecular weights. The presence of two molecular forms of ChE was also demonstrated in the brain of the rat (Fig. 2d). In rat retina, however, only one form could be clearly demonstrated (Fig. 2e). This form was found to possess the same S value and mol. wt as the major activity form of the brain. Also in guinea pig iris the ChE could be separated in a higher and lower mol. wt form by sucrose gradient centrifugation (Fig. 2g).

The proportion between the higher and lower mol. wt form in chicken brain and retina was demonstrated to differ in such a way that the lower molecular weight form seemed to predominate during the early recovery stages after inhibition by soman (Fig. 2a, b). For rat brain and retina ChE, however, the higher mol. wt form was always solubilized in the highest proportion (Fig. 2d,e). In the case of the guinea pig iris the higher mol. wt form was also always solubilized in the highest proportion during the ChE recovery stages following soman inhibition (Fig. 2g).

Administration of L-anserine nitrate, L-homocarnosine sulphate, L-carnosine and thyrotropin releasing hormone to the rat and primobolan to the mouse was not found to affect the recovery of activity and molecular forms of ChE following irreversible ChE inhibition. In the rat hypophysectomy was not found to affect the recovery either.

Previously Andersen & Mikalsen (1978a) have studied the polyacrylamide gel electrophoresis patterns of brain, retina and serum ChE from chicken and rat. The general band pattern as shown in their work was

not found to change during ChE recovery neither following inhibition by DFP nor by soman.

Use of PrThChI and BuThChCI as substrates in the activity measurements of fractions collected after sucrose gradient centrifugation as well as in the staining procedure of the polyacrylamide gels did not reveal the presence of other ChE enzymes than those already indicated.

DISCUSSION

Previous studies by Yaksh et al. (1975) showed the t~ value for ChE recovery in the cerebral cortex of the cat to be 3.9 days following soman inhibition. It appears from their results that the ChE of the cerebral cortex exhibited longer recovery time compared to ChE of other brain parts investigated. In addition the t~ recovery times of ChE of rat and guinea pig whole brain homogenates were found to be 2.3 and 4.7 days, respectively. Our values for the rat, however, were higher. For cerebral cortex the t~ values for ChE recovery were 13 days for females and 12 days for males. Glow et al. (1966) found t~ values of 7-11 days for recovery of ChE in various parts of the rat brain, while Austin & James (1970) found a t~ value of 16.5 days for recovery of ChE in rat cerebral cortex after inhibition by DFP. These results are in coincidence with the t, value of 12 days found in the present work for ChE of the mouse brain after DFP inhibition. Our results thus do not indicate significant differences between soman and DFP inhibition with respect to ChE recovery rates. Such a difference, however, seem to be indicated by the work of Filbert et al. (1972) and Yaksh et al. (1975). In general, protein turnover in brain tissue is thought to be about 10-20 days (von Hungen et al., 1968; Lajtha et al., 1971). Our work therefore do not seem to indicate a different turnover of brain ChE compared to the general turnover of other proteins in brain tissue.

Yaksh et al. (1975) showed that in subcellular fractions of cat brain tissue the soluble form of ChE exhibited a shorter t~ value of recovery after soman inhibition than the membrane bound form in microsomes and synaptosomes. In resemblance our results for the rat show a shorter recovery value for the soluble plasma ChE after soman inhibition than the membrane bound forms predominating in erythrocytes and brain. Similar results were found for the mouse and the chicken (see Table 3). The recovery rate of the soluble ChE in the cat cerebrospinal fluid (CSF) fol-


lowing soman inhibition seem to be comparable to that of blood plasma ChE, i.e. faster than for membrane bound ChE (Yaksh et al., 1975). This may, however, not be surprising because the CSF itself is de- rived from the blood plasma in the choroid plexus (Davson, 1967).

After irreversible ChE inhibition of the frog by the compound 217 AO the original level of acetylcholine sensitive ChE (AcChE) in the central nervous system was restored in 30 days while the butyrylcholine sensitive ChE (BuChE) in brain and nerve was restored in 7 days (Clouet & Waelsch, 1961). These authors, however, did not investigate whether these two ChE forms occur as soluble or membrane bound in the frog. Generally in the animal kingdom AChEs are membrane bound, while BuChEs occur in soluble form (Silver, 1974).

The total plasma ChE of the rat exhibited the phenomenon of overshoot, i.e. the ChE activity reached a level higher than the original level after soman inhibition. The overshoot effect was also found to occur in the liver of rats and mice following ChE inhibition by soman and DFP, respectively. This may not be surprising as plasma is produced in the liver. Domschke et al. (1970) also demonstrated ChE overshoot in the liver of male rats following soman inhibition. This phenomenon has otherwise been described for plasma ChE of the blue fox (S~li et al., 1977) after inhibition by fenchlorphos and in plasma ChE of the guinea pig inhibited by soman (Laake & Smith, 1975) Our results did not show such an effect in the chicken. It appears from the work of Clouet & Waelsch (1961) that a BuCh sensitive ChE in the blood of the frog showed a significant overshoot following inhibition by the irreversible inhibitor 217 AO. The overshoot effect therefore do not seem to be con- fined only to mammalian species. In fact not all mammalian species seem to exhibit this phenomenon either. A closer study of the work of Yaksh et al. (1975) did not indicate ChE overshoot to occur in the cat blood plasma after soman inhibition. Long term studies of this phenomenon were not performed in the present work, but in the experiments of Soli et al. (1977), the plasma ChE was found higher than the normal level until 120 days after inhibition.

The observation that brain ChE from various species is composed of two molecular forms, is in accord- ance with the results of other workers (Rieger & Vigny, 1976; Rieger et al., 1976; Henderson, 1977; Marchand et al., 1977; Andersen & Mikalsen, 1978b, 1979). Our results, however, indicate that the proportions between the two forms in cortex and retina may vary. Similar differences in activity proportions have been demonstrated previously for ChE molecular forms in the brain and retina of the frog (Andersen & Mikalsen, 1979). It is interesting to view the differences in activity proportions on the background of the retina being embryologically a part of the brain. Based on sucrose gradient centrifugation the soluble plasma ChE of chicken and rat was not found to be composed of more than one isoenzyme form.

In a series of experiments Andersen & Mikalsen (1978b) studied the influence by different concen- trations of the detergent Triton X-100 on solubilization of chicken cortex ChE. It appeared from these experiments that the heavier ChE form always was

solubilized in the highest proportion regardless of the detergent concentration. The lighter ChE form is, however, solubilized in the highest proportion during the beginning of the ChE recovery time following soman inhibition of the chicken. At later stages the heavier ChE form predominates. The retina showed similarity to the brain in these respects. In our opinion this strongly suggests that the lighter ChE form is the first one to be synthesized after irreversible ChE inhibition. In the case of the rat, however, only the heavier ChE form could be solubilized from cortex in the highest proportion by means of the optimal solubilizing concentration of 0.25% Triton X-100. Rieger & Vigny (1976), who studied the solubilisation of ChE from rat brain tissue by a procedure comparable to that used in the present work, were also able to separate two ChE forms on sucrose gradients. These forms seemed to be identical to those found in the present work with regard to S values. When the authors used EDTA or saline buffer for extraction of membrane bound ChE, the activity of the lighter ChE form was found in a higher proportion. This solubilisation procedure must be considered a rather lenient treatment compared to the use of Triton X-100. This may indicate that the rat ChE may be susceptible to aggregation in some way by the influence of Triton X-100. The present results may therefore be interpreted in such a way that the ChE initially formed after irreversible ChE inhibition nevertheless exists mainly in the lighter form.

The development and maturation into functional forms of ChE following conception have previously been studied in chicken brain by Marchand et al. (1977) and in rat brain by Rieger & Vigny (1976). These authors found the lighter ChE form to be present in higher proportions in earlier stages for both species. Studies of various cell and tissue cultures also indicate that the pattern of ChE isozymes progressively shifted from a preponderance of lower to higher mol. wt forms following irreversible ChE inhibition (Wilson & Walker, 1974; Rieger et al., 1976). These results compared to those of the present work may therefore indicate a general mechanism by which ChE is synthesized and incorporated into its functional membrane form in such a way that this form is preceded by a form of lower mol. wt. This form may be freely present in the plasma and more easily extractable in earlier stages in the membrane incorporating process. It appears from the data in the present work both for the chicken and rat that the normally present soluble blood plasma ChE is not identical to this precursor form, This can be deduced from the differences in sedimentation constants observed for the ChE forms. Substrate specificities and inhibition constants of the blood plasma ChE and the membrane bound ChE forms are also different. This is shown by data given by Andersen & Mikalsen (1978a, b).

Recovery times for ChE following irreversible ChE inhibition in cell and tissue cultures seem generally considerably shorter than those observed for ChE in intact animals. Rieger et al. (1976) found de novo synthesis of ChE in mouse neuroblastoma cells to be nearly completed in 24 hr, while in chicken embryo muscle cultures Wilson & Walker (1974) found that 78% of the activity returned within 4 hr. Such differ-

c.n.p. 7 1 / 1 ~ c


ences in recovery rates for ChE between cultures and whole animals might indicate tha t the regulation of ChE in the body do not only reside within the ChE producing cells themselves. The ChE recovery studied in the present work was not, however, found to be influenced by hypophysectomy, p r imobolan or by some peptides. Harris et al. (1974) found evidence indicating that N6,O2-dibutyryl adenosine Y,5'-cyclic monophospha te (DBcAMP) and c A M P may be involved in the regulation of ChE synthesis.

Acknowledgement--The skilful technical assistance of Mrs Lily Wang, Mrs May-Britt Nordkild Aas and Mr Tore Saetre is very much appreciated.

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