Zeitschrift ffir Zellforschung 85, 264 270 (1968)

The Uhrastructural Localization of a Cholinesterase in the Body Muscle of Plaice (Pleuronectes platessa)

JOHAN LUNDIN and BIRGITTA HELLSTROM *

Research Institute of National Defence, Department 1, Sundbyberg 4, and The Department of Anatomy and Histology, and The Laboratory for Electron Microscopy, Royal Veterinary

College, Stockholm, Sweden

Received October 25, 1967

Summary. By means of a histochemical method adapted for electron microscopy a cholin- esterase in body muscle cells of plaice (Pleuronectes platessa) has been localized to the sarcolemma. The cholinesterase activity disappeared from the sarcolemma after the muscle tissue had been incubated with a bacterial enzyme, which had earlier been shown, by bio- chemical methods, to be able to liberate this cholinesterase activity from plaice muscle.

Pre l iminary histochemical exper iments with l ight microscopy indicated tha t

a cholinesterase ac t iv i ty in plaice body muscles was localized to the muscle cell

surface (Lu~DIN, 1962). I t has been found tha t this f i rmly s t ruc ture-bound cholin-

esterase could be l iberated and solubilized by means of a bacter ial enzyme

(LvNDIN and BOVALLIVS, 1966; LUNDIN, 1967). Biochemical studies have shown

tha t the solubilized cholinesterase splits both acetylcholine and butyrylchol ine

(LuNDIN, 1967). The present invest igat ion was under taken in order to establish

the cytological localization of the cholinesterase ac t iv i ty and to f ind out if this

ac t iv i ty disappeared or diminished after t r e a t m e n t wi th the bacter ial enzyme.

KAR~OVSKY'S (1964) thiocholine method for electron microscopy was used. This

method is based on the fact t h a t cholinesterase splits acetyl thiochol ine (and

butyryl thiocholine) . The thiocholine formed, reduces ferr icyanide to ferrocyanide,

which forms electron-opaque deposits of copper ferrocyanide.

Materials and Methods Tissues. Plaice (Pleuronectes platessa) was caught at Kristineberg Zoological Station and

sent alive to the laboratory, where they were handled in a coldroom. The fish was killed by decapitation. The skin of their upper side was removed and small (1--5 mm a) muscle pieces from the immediate vicinity of the spine were dissected out and placed in cold ( 2 ~ ~ C) isotonic sucrose (Mallinckrodt Chemical Works, St. Louis, U.S.A.) solution.

Liberation o/ Cholinesterase Activity. The cholinesterase activity was liberated from the cell structures by an enzyme obtained from the growth medium of a Cytophaga sp. (Lu~- nI•, 1967). The activity of the bacterial enzyme was tested as described elsewhere (LuNmN, 1967). The plaice muscle pieces were incubated for two hours at 30~ in isotonic sucrose solutions with or without the bacterial enzyme. Control tests, in which the specimens were kept at 0 ~ C in the same medium without bacterial enzyme, were also performed. All muscle pieces were then fixed for two hours at 0 ~ C in 4% formaldehyde in isotonic sucrose solution main- tained at pH 7.6 by 0.075 M phosphate buffer (KARNOVSKY, 1964). After fixation, the muscle pieces were kept overnight in isotonic sucrose solution at 0 ~ C. They contained 60 % of the total cholinesterase activity as compared biochemically with unfixed controls. For a discus- sion of the probable effects of fixation, see e.g. BRZIN et al. (1966).

* The provision of live plaices from Kristineberg Zoological Station, Fiskeb/~ckskil, Swe- den, is gratefully acknowledged. We are greatly indebted to Dr. AKE BOVALLIUS, FOA, who provided the starting material for the bacterial enzyme. We would like to express our sincere thanks to Prof. LENNART NICANDER for valuable discussions and for placing the resources of the Department of Anatomy and Histology, Royal Veterinary College, to our disposal.

Localization of a Cholinesterase in Plaice Muscle 265

Demonstration o/ Cholinssterase Activity. The fixed muscle pieces were incubated with the cholinesterase substrates without further sectioning (KARNOVSKY, 1964) since they would have been impossible to handle, if they had been frozen and cut into thinner sections. Incuba- tion with the cholinesterase substrates acetylthiocholine and butyrylthiocholine bromide, 2 x 10 -a M, was performed according to KARZCOVSKY (1964) for 45 minutes at pH 6 and 0 ~ C. The localization of the cholinesterase activity was controlled in two ways: a) By inhibiting the enzyme, the precipitates resulting from the activity should disappear. Inhibition of the cholinesterase activity was accomplished with 10 -s M DFP (di-isopropyl-fluorophosphate). In these experiments, the formaldehyde-fixed muscle pieces were preincubated with the inhi- bitor for one hour before incubation with the substrate solution, which also contained the same concentration of the inhibitor, b) Precipitates resulting from unspecific deposits of copper compounds should appear even ff no substrate was present in the incubation medium. These controls were performed accordingly.

Electron Microscopy. After completion of the histochemical procedure, the material was fixed for one hour in chilled, buffered 2% osmium tetroxide solution (MILLONIO, 1962), and then dehydrated and embedded in Epon (LEFT, 1961). Thin sections (LKB Ultrotome) from the surfaces of the embedded pieces were stained first with lead acetate for two hours (DAL- TOn and ZEICEL, 1960) and subsequently with uranyl acetate for 15 minutes (WATson, 1958). The sections were examined in a Siemens Elmiskop I at 60 kV.

Results 1

Cholinesterase a c t i v i t y was d e m o n s t r a t e d in sa rco lemmal s t ruc tures of plaice muscle cells when using bo th acetyt th iochol ine and bn ty ry l th iocho l ine as sub- s t ra tes (Figs. 1, 4). I n the concent ra t ions used the two subs t ra tes resul ted in comparab le amoun t s of e lec t ron-opaque precip i ta tes . E n z y m e ac t i v i t y was also d e m o n s t r a t e d in ex t race l lu la r ly loca ted membranes (Fig. 6). I n these membranes , the si tes of a c t i v i t y seem to be sepa ra t ed b y regular in tervals . P rec ip i t a tes occasional ly occurred between the inner and outer membranes of mi tochondr ia (Figs. 1, 4, 6).

The cholinesterase inh ib i tor D F P , 10 -6 M, abol ished all p rec ip i ta tes except poss ib ly a round some mi tochondr ia (Fig. 2). The muscle pieces i ncuba ted in media , which d id no t conta in a subs t ra te , showed no prec ip i ta tes loca ted to the sarcolemma. Occasional ly some prec ip i ta tes occurred a round the mi tochondr ia (Fig. 3).

W h e n the muscle pieces were i ncuba ted a t 30 ~ C for two hours wi th a puri- fied bac te r ia l enzyme the sarcolemmal prec ip i ta tes d i sappeared (Fig. 5), demon- s t ra t ing t h a t the t r e a t m e n t had removed the cholinesterase a c t i v i t y f rom these membranes .

Morphological changes, unequivoca l ly due to the ac t ion of the cholinesterase l ibera t ing enzs-me , could no t be demons t r a t ed in the p resen t inves t iga t ion . Some- t imes the sarco lemma seemed p a r t l y to have d i sappeared or have been d i s rup ted (Fig. 5). The incuba t ion wi thou t bac te r ia l enzyme a t 30~ for two hours d id no t affect the local izat ion of the cholinesterase ac t iv i ty .

Discussion The observed ex t race l lu la r membranes , d i sp lay ing regular ly spaced sites of

chol inesterase ac t i v i t y (Fig. 6), m a y be blebs or s t r ipped off pa r t s of the sarco- l emma t h a t have curled together , as the same regular a r r angemen t of ac t i v i t y can somet imes be observed also on in t ac t sa rco lemma (Figs. l , 6). They m a y also be axon membranes , since postgangl ionic adrenergie nerves in mouse hea r t

1 List o/ Abbreviations. CF Collagen fibers; EM Extraeellular membrane; M Mitoehon- drion; SL Sarcolemma; SR Sarcoplasmie retieulum.

266 J. LUNDIN and B. HELLSTROM:

Fig. 1. Normal cholinesterase activity. Oblique section of a plaice muscle cell kept at 0 ~ C in isotonic sucrose solution showing black precipitates along the sarcolemma as a result of cholinesterase activity. Substrate : acetylthiocholine for 45 minutes at 0 ~ C. Small amounts of precipitate can be discerned between the inner and outer membrane of one mitochondrion

to the right. Magnification 21000 •

Fig. 2. Inhibition of cholinesterase activity by DFP. Oblique section through two plaice muscle cells treated as described in the legend to Fig. 1 but, in addition, in the presence of

Localization of a Cholinesterase in Plaice Muscle 267

were recently shown by the same method as the present one (KARNOVSKY, 1964), to contain acetylcholinesterase similarly located (GRAF, 1967). The localization of acetylcholinesterase in (frog) axonal membranes has also been demonstrated e.g. by BRzI• (1966) by a similar histochemical method. In any case, the regular spacing of the enzymic sites may offer interesting implications in respect to the physiological function of cholinesterase. Thus NAC~MANSOH~ (1966) has claimed a role for acetylcholine in the permeability cycle of excitable membranes, and ac- cording to his theory cholinesterase should be present all along such membranes. The reference cited gives a recent review of this subject.

The precipitates, occasionally occurring between the inner and outer mem- branes of mitochondria (Figs. 1, 4, 6), were observed in unfixed tissue also by KARNOVSKY (1964) and are probably not due to cholinesterase. This is obvious since these localizations of precipitates occur both in preparations in which the cholinesterase activity was inhibited by D F P and in controls incubated in the absence of substrate (Figs. 2, 3).

During preliminary experiments, it was noticed tha t the success of the method for the plaice muscle used was highly dependent on the pH in the substrate incubation medium. When the pH was too high (above 7), several additional localizations of precipitates were obtained, which could occur irrespective of the presence of snbstrates, DFP or bacterial enzyme. Several of these localizations were described for cardiac muscle by KARNOVSXY (1964) and also by other authors (KoELLE and FOROGLOu-KERAMEOS, 1965; ULBRECHT and KRUCKEN- BERG, 1965) working at the optimal pH 6. Thus precipitates were found in the A-bands, in the sarcoplasmic reticulum, around the membranes of the nuclei, and at collagen threads. The latter two types have been regarded as artefacts (KARNOVSKY, 1964; KOELLE and FOROGLGU-KERAMEOS, 1965). Concerning the A-band precipitates in ra t cardiac muscle, KARNOVSKY (1964) finds their locali- zation indicative of the presence of myosincholinesterase activity (KSvb.R and Kov~.cs, 1957). In the present investigation, the irregularly appearing precipi- tates in the A-bands cannot be taken as evidence of the existence of myosincho- linesterase in the plaice body muscles, although cholinesterase activity has been found in myosin fractions prepared from another fish, Amiurus nebulosus, by KSV~R et al. (1963) and SZABOLCS et al. (1963). The localization of the precipi- tates on the A-bands seems to be related to the contraction state of the muscle fiber, which may prompt an allusion to work done e.g. by T:CE and BARR- :NETT (1962) to localize ATP-ase activity in heart muscle myofibrils of ra t by means of lead phosphate formed in the presence of ATP and a lead salt. This enzyme is located in a remarkably similar way in different parts of the A-bands, as described by KARNOVSKu (1964) for the proposed myosincholinesterase and as has also been noted on occasion by the present authors (see above). I t is pos- sible tha t unspecific localizations of heavy metal precipitates may appear more

10 -6 M DFP. Substrate: acetylthiocholine. Collagen threads can be observed. No precipitates are located to the sarcolemma. Some precipitate has formed in the external membranes of

one mitochondrion. Magnification 8400 •

Fig. 3. Control. Oblique section of two partially separated plaice muscle cells treated as described in the legend to Fig. 1 apart from the absence of any substrate in the incubation medium. No precipitates have appeared in the sarcolemma. Some mitochondria display

precipitates between the inner and outer membranes. Magnification 8400 •

268 J. LUNDIN and B. HELLSTRSM:

Fig. 4. Normal cholinesterase activity. Oblique section of two partially separated plaice muscle cells. Treatment as described in the legend to Fig. 1. Substrate: butyrylthiocholine. The same results are obtained as with acetylthiocholine shown in Fig. 1. Magnification 14000 X

Fig. 5. Enzymic removal of cholinesterase activity. Transverse section through plaice muscle cell incubated at 30 ~ C for two hours with a cholinesterase liberating enzyme from the bac- terial strain Cytophaga sp. in isotonic sucrose solution. Substrate: butyrylthiocholine for 45 minutes at 0 ~ C. The sarcolemma is devoid of precipitate and does not seem to be intact

(cf. Fig. 1). Magnification 34000 x

o f t en t h a n is u n r a v e l l e d e m p l o y i n g p r e sen t c y t o c h e m i c a l m e t h o d s for chol in-

es terase . Th is m a y be t h e case in t h e p resence of SH-g roups , in pa r t i cu la r , wh ich are a b u n d a n t in m y o s i n (TIc~ a n d BARRNETT, 1962). F u r t h e r s tud ies a re r e q u i r e d

Localization of a Cholinesterase in Plaice Muscle 269

Fig. 6. Normal cholinesterase activity. Oblique section through plaice muscle cell and extra- cellular membranes. Substrate: butyrylthiocholine. The electron-opaque precipitates seem

to be regularly spaced. Magnification 23000 •

to elucidate the question of the cytochemical demonstra t ion of cholinesterase act ivi ty in the A-band.

The sarcoplasmic ret iculum (in this work denoting both the transversal and longitudinal tubular systems) in the plaice muscle cells does not seem to contain cholinesterase activity. KAR~OVSKu (1964) found cholinesterase act ivi ty in the

270 J. LUNDIN and B. HELLSTR61~I: Localization of a Cholinesterase in Plaice Muscle

longi tudinal system in the ra t cardiac muscle cells. Acetylcholinesterase has also been demonst ra ted in the sarcoplasmic re t iculum of rabb i t psoas muscle by means of another histochemical technique (ULBR]~CHT and KRUCKE•BERG, 1965). The different results may depend on the different materials and techniques employed. The electron dense mater ial regularly found in the tubules, f requent ly appearing as very dense, rounded forms (Figs. 1--3), was found also in mater ial tha t was no t incuba ted in the substrate medium. According to several authors (for a review, see SMITH, 1966) calcium complexes might be the cause of such electron dense material .

R e f e r e n c e s

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DALTON, •. J., and R. F. ZEIGEL: A simplified method of staining thin sections of biological material with lead hydroxide for electron microscopy. J. biophys, biochem. Cytol. 7, 409 (1960).

G~AF, J. : Elektronenmikroskopischer Nachweis der Acctylcholinesterase in den postganglio- n~ren sympathischen Nerven des Herzens. J. Neurochem. 14, 893--897 (1967).

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KOELLE, G. B., and C. FOROGLOU-KERAMEOS: Electron microscopic localization of cholin- esterases in a sympathetic ganglion by a gold-thiolacetic acid method. Life Sci. 4, 417--424 (1965).

KSV~R, A., and T. KovXcs: On the specificity of myosincholinesterase. Acta physiol. Acad. Sci. hung. 11, 259--265 (1957).

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TICE, L.W., and R. J. BARRNETT: Fine structural localization of adenosinetriphosphatase activity in heart muscle myofibrils. J. Cell Biol. 15, 4 0 1 ~ 1 6 (1962).

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Dr. JOHAN LU~DI~r Research Institute of National Defence Department 1 Sundbyberg 4, Sweden