Choline acetylase and cholinesterase activity in denervated electroplax

266

BIOCHIMICA ET BIOPHYSICA ACTA BBA 4 1 9 9

CHOLINE ACETYLASE AND CHOLINESTERASE DENERVATED

ACTIVITY IN

ELECTROPLAX

P H I L I P R O S E N B E R G , E D M U N D A. MACKEY*, H E N R Y B. H1GMAN** AND WOLF-D. D E T T B A R N

Departments of Biochemistry and Neurology, and Department of Pathology, Division of Neuropathology, College of Physicians and Surgeons, Columbia University, New York, N. Y. (U.S.A.) (Received May 6th, 1963)

SUMMARY

I. Following denervation of the Sachs electric organ of Electrophorus, choline acetylase (acetyl-CoA: choline 0-acetyl transferase, EC 2.3.1.6) activity decreased about 50 % in one week and 9 ° % in four weeks, no activity was detectable after 74-97 days. In the Main electric organ 97 days after denervation choline acetylase activity is less than 1 % that of control tissue. The medium for testing choline acetylase activity was improved by varying some of the components. 2. Tested 78 days after denervation cholinesterase activity of the Sachs organ was not significantly decreased. 3. Structural changes were microscopically detectable in the terminal neural innervation of the electroplax as early as 2 days following denervation, while after 2o days there was a virtual absence of nerve fibers. 4. The changes in electrical activity of the denervated cells are discussed in reference to the changes in structure and enzyme activities. INTRODUCTION

During degeneration of a nerve fiber, as shown by NACHMANSOHN and associates, conduction is blocked when one third of the choline acetylase (acetyl-CoA: choline O-acetyl transferase, EC 2.3.1.6) activity is still present ; the enzyme activity continues to fall, reaching a very low level within one week following section 1. Since then other investigators also have found that the choline aeetylase activity in nerve tissue falls to very low levels within one week following denervation 2, ~. In contrast, acetylcholinesterase (acetyl choline acetyl-hydrolase, EC 3.1.1.7) activity in degenerating nerve only decreases about 60 % in one week, and the level then remains constant for several more weeks 4. Following section of the preganglionic fibers of the superior cervical ganglia, a loss in acetylcholine, choline acetylase and acetylcholinesterase activity is observed 2,54 As with degenerating nerve the choline acetylase activity falls more abruptly and to a lower level than the aeetylcholinesterase activity. Decreases in acetylcholinesterase * Present address: Dept. of Pathology, University of Miami, Miami, Fla. (U.S.A.). ** Present address: Dept. of P s y c h i a t r y a n d Neurology, Louisiana State University, School of Medicine, New Orleans, La. (U.S.A.).

Biochim. Biophys. Acta, 8z (1964) 266-275

ENZYMIC ACTIVITY IN DENERVATED ELECTROPLAX

2t)7

activity following denervation have also been observed in striated muscleg, 1° and spinal cord 11. No systematic studies have been performed with denervated electric tissue even though electric organs were of decisive value in m a n y studies exploring the role of acetylcholine in bioelectrogenesis 12. It has been reported that following denervation the indirect response of the electroplax from the electric organ of the Electrophorus is rapidly lost whereas a response of the cells m a y be directly elicited by stimulation for long periods of timO T M . In two of these studies, however, no microscopic control was presented to show that the cells were completely denervated~4,15. It appeared of interest therefore to measure choline acetylase and acetylcholinesterase activities in electroplax following denervation under the control of microscopic studies. MATERIALS AND METHODS

The Sachs and Main electric organs of the electric eel (Electrophorus electricus) were denervated b y destruction of an appropriate length of spinal cord.

Measurement of acetylcholinesterase activity Homogenized normal or denervated tissue taken from the organ of Sachs was incubated at room temperature for I or 2 h in a specially modified Ringer's solution TM buffered at p H 7.6-7.7 with o.I M Tris. This solution also contained either I, 5 or 25 mM acetylcholine bromide as substrate. Enzyme activity was determined by the HESTRIN colorimetric technique aT.

Preparation and assay of phosphotransacetylase Phosphotransacetylase (acetyl-CoA : orthophosphate acetyltransferase, EC 2.3.1.8) was prepared from Escherichia coli No. 4157 obtained from American Type Culture Collection 2II2M ST., N.W., Washington, D.C., using a medium similar to that described b y UMBREIT AND GUNSALUS1 . Following harvesting, the cells were lyophilized, ground with alumina and 0.02 M potassium phosphate buffer (pH 7) and the supernatant was extracted with ammonium sulfate TM. The final preparations had activities of 26-40 units per ml when assayed by the method of STADTMAN20.

Choline acetylase activity measurements The method for determination of choline acetylase activity was modified from that of BERMAN, WILSON AND NACHMANSOHN21. In preliminary experiments the incubation mixture differed from that finally used by containing no potassium chloride, half the amounts of choline and CoA and dilithium rather than dipotassium acetyl phosphate. In this medium, used previously, normal Main organ synthesized only about 15o #g acetylcholine per gram per hour which is only about half the value previously found 22. Increasing the activity of phosphotransacetylase in the media did not alter the activity observed nor did it make any difference whether the tissue was homogenized at room temperature, 3 ° or - - I O °. CoA, 75 % pure, was obtained from Mann Laboratories. Dipotassium acetyl phosphate was prepared by mixing 16o mg dilithium acetyl phosphate dissolved in 1.8 ml distilled water with I g of washed Amberlite IR-I2O resin. This mixture was filtered Biochim. Biophys. Acta, 82 (1964) 266-275

268

P. ROSENBERG et al.

and pH of the filtrate adjusted to 7.0 with IO M potassium hydroxide. The volume was made up to 2 ml with water. The composition of the incubation mixture (final volume o.5 ml) which was used in the experiments reported in this paper, and which seemed to give the highest values for choline acetylase activity was as follows: CoA, o.5 /~mole/ml, o.2 ml; dipotassium acetyl phosphate (see above) o.o8 ml; phosphotransacetylase, ~ 3o units/ml, o.o5 ml; choline chloride, 5oo/~moles/ml, o.o 4 ml ; tetraethytpyrophosphate, 5o/~moles/ml, o.o2 lnl; magnesium chloride, 25o /xmoles/ml, o.o2 ml; L-cysteine hydrochloride (neutralized), 950 /~moles/ml, 0.04 ml; potassium phosphate buffer (pH 7), 500 /mloles/ml, o.o2 ml; potassium chloride, 133o /,moles/ml, o.o 3 ml. This mixture was incubated at room temperature for 15 min to allow formation of acetyl CoA. Either o.I g of homogenized electric tissue plus o. 4 ml of water or o.5 g of electric tissue was added and incubation proceeded for 2o or 6o rain depending on enzyme activitv. Control tubes contained the same material except that the tissue had been heated to destroy choline acetvlase activity. At the end of the incubation period ml of o.Io N HCI was added to the I ml of incubate. Volume was made up to 50 ml with frog Ringer's of the following composition; I19 mM NaC1, 2.5 mM KC1, 1.8 mM CaCl2 and 2.5 mM NaHCOa, to which was added sufficient dextrose and physostigmine salicylate to give final concentrations of 4 mM and o.o24 mM respectively. The acetylcholine content of dilutions of the above incubate were determined by bioassay using a frog rectus abdominis muscle. Isometric contractions were recorded with the aid of an RCA transducer tube (5734) on a Varian ink writer. Contractions by the experimental solutions were tested against 5' IO-S to 2" IO -7 M acetylcholine solutions depending on the sensitivity of the preparation. The control mixture containing heated tissue gave no contraction. Histolog 3' Biopsy specimens were obtained from the organ of Sachs and were fixed in a IO % neutral formalin saline solution. Tissue blocks were embedded in paraffin, and sections were stained by hematoxylin-eosin, the Mahon stain for myelin sheaths, and by the GLEES method for axons 2a. Following examination of the electrical activity of isolated single electroplax of the normal, and of the denervated, Sachs organ, the individual electroplax were treated in the same way. Frozen sections were also prepared and stained with the Bielschowsky silver method for axons. Intravital staining of terminal nerve fibers of Sachs organ was carried out at the time of biopsy. For this stain a o.o5 % solution of methylene blue was used. Staining and preparation of tissue sections were done according to the method of CoRRs AND WOOLE24. Biopsy material was thus prepared from normal control animals, and from animals, at varying intervals of 2-97 days, after destruction of the spinal cord. RESULTS

Structural fincCings The eleetroplax of Sachs organ, as is well known 2~-2s, receive their innervation exclusively by way of the small papillae on the caudal surface of the electroplax. The larger rostral papillae receive no direct nerve supply. Near the papillae the terminal nerve fibers branch and form an intricate plexus on the surface of the papillae (Figs. i and 2). Biochim. Bioph),s. Acla, 82 (t9',4) 260-275

ENZYMIC ACTIVITY IN DENERVATED ELECTROPLAX

269

Fig. i. T e r m i n a l n e r v e fibers i n n e r v a t i n g c a u d a l papillae of electroplax. I n n e r v a t i o n p a t t e r n in n o r m a l control animal. F r o z e n section. M e t h y l e n e blue s t a i n ; × 72,5.

Structural changes were detectable in the terminal nerve fibers as early as 2 days after destruction of the spinal cord. When stained b y methylene blue, or by silverimpregnation methods, m a n y of the terminal nerve fibers showed bulbous swellings along their course through the caudal chamber, and m a n y of these ended in similar irregular swellings near the caudal papillae. Many papillae were devoid of terminal plexuses. Many of the terminal nerve fibers also showed diffuse swelling and focal fragmentation (Fig. 3). At this stage the larger nerve fibers, and nerve fascicles, were histologically normal. The terminal nerve fibers and terminal plexuses were rapidly lost following destruction of the spinal cord. No normal terminal innervation pattern was identified in material examined from IO to 80 days following the cord lesions (Fig. 4). In the tissue denervated for a period of 20-80 days, there was virtual absence of small nerve fibers near the electroplax and in the gelatinous matrix of their caudal chambers. Large nerve fascicles in the connective tissue septae showed a striking reduction in number Biochim. Biophys. Acla, 82 (1964) 266 275

27 °

p. ROSENBERG et al.

Fig. 2. Branching nerve fibers in caudal chamber and around caudal papillae. Innervation pattern in normal control animal. Paraffin section. Glees silver method; × 725 .

of nerve fibers, in the later stages of denervation. The fibers r e m a i n i n g in these n e r v e b u n d l e s m a y have been sensory, or post-ganglionic autonomic, nerves. The results of e x a m i n a t i o n of the single electroplax were similar to those o b t a i n e d in the biopsy material. There was rapid disappearance of t e r m i n a l nerve fibers, a n d d e n e r v a t i o n of the caudal chambers a n d papillae appeared to be complete after 20 days.

Choline acetylase measurements To test the reliability of our assay 2 0 / , g of acetylcholine were added to i n c u b a t i o n m i x t u r e s c o n t a i n i n g n o r m a l or d e n e r v a t e d tissue of Sachs organ. The recovery according to the bioassay was 18 a n d 21/~g respectively. I n two experiments in which 2 ~g of acetylcholine were added to d e n e r v a t e d tissue of Sachs organ 2. 7 a n d 2.4/~g were recovered. To test w h e t h e r in the d e n e r v a t e d tissue of Main or Sachs organ an inhibitor of choline acetylase m a y have been present, the a c t i v i t y of k n o w n a m o u n t s of n o r m a l a n d d e n e r v a t e d electric tissue m i x e d betore or after i n c u b a t i o n was determined. The tissue d e n e r v a t e d for a long time (97 days), h a d no measurable activity,

Biochim. I3iophys. Acla,

82

(~964) 266-275

ENZYMIC ACTIVITY IN DENERVATED ELECTROPLAX

271

Fig. 3. Swelling and fragmentation of terminal nerve fibers in caudal chamber. Destruction of spinal cord 2 days previously. Frozen section. Methylene blue stain; × 725 .

whereas the c o m b i n a t i o n of n o r m a l plus d e n e r v a t e d tissue s y n t h e s i z e d 4 - 6 / ~ g more acetylcholine per g r a m per h o u r t h a n n o r m a l tissue alone. Since only two e x p e r i m e n t s were p e r f o r m e d the question r e m a i n s w h e t h e r this increase in a c t i v i t y is significant. No evidence of a n y inhibition of choline a c e t y l a s e of n o r m a l tissue bv the d e n e r v a t e d tissue was o b t a i n e d . The choline acetylase a c t i v i t y of the Sachs organ of a n o r m a l eel (6 experiments) a n d t h a t of the s a m e eel 2, 6, IO a n d 25 d a y s following d e n e r v a t i o n was tested. The acetylcholine synthesis prior to d e n e r v a t i o n was 6o i 2/~g per g r a m fresh tissue p e r hour. The decrease in a c t i v i t y as a function of d a y s d e n e r v a t e d is shown in Fig. 5. The d a t a after 2 d a y s are b a s e d on a single d e t e r m i n a t i o n , t h a t a t t e r 6 a n d IO d a y s upon duplicate, t h a t after 25 d a y s u p o n three d e t e r m i n a t i o n s . The tissue of Sachs o r g a n of several o t h e r eels showed no d e t e c t a b l e choline a c e t y l a s e a c t i v i t y 74-97 d a y s following d e n e r v a t i o n , i n d i c a t i n g a c t i v i t y less t h a n 1.5 ~g acetylcholine s y n t h e s i z e d per g r a m p e r h o u r which is the limit of the a s s a y m e t h o d s used. I n two e x p e r i m e n t s tissue of t h e Main organ of an eel which h a d been d e n e r v a t e d 97 d a y s s y n t h e s i z e d 2 / , g of acetylBiochim. Biophys. Acta, 82 (i964) 266 275

272

P. ROSENBERG g~ a[.

choline per gram per hour. In the experiments with normal tissue of Main organ the activity ranged from 320 to 460 tzg acetylcholine synthesized per gram per hour.

[qg- 4. C a u d a l p a p i l l a e of e l e c t r o p l a x and c a u d a l c h a m b e r de voi d of t e r m i n a l ne rve libcrs, l)es t r u c t i o n of spinal cord 72 d a y s p r e v i o u s l y . Paraffin section. (;lees silver m e t h o d : ~ 725.

("holinesterase activi(v The data on cholinesterase (acylcholine acyl-hydrolase, EC 3.1.1.8) activity of Sachs organ from normal and denervated electric eels are shown in Table I. Although only two experiments were performed at each concentration of acetylcholine, it appears that no significant decrease in activity occurred following denervation. The electrical activity of isolated single electroplax of the denervated Sachs organ was checked by Mr. T. R. PODLESKIand Miss E. BARTELS. Response to indirect stimulation by way of nerve endings was lost very quickly (in about 2 days), whereas the directly elicited response IO days following denervation was apparently normal. From 25 to 97 days following denervation the direct response was usually small, graded and non conducted, although sometimes an apparently normal full-size conducted direct response was obtained. Biochin~. ]~inphy~..4c/a, 82 (1964) 2(~ 275

273

ENZYMIC ACTIVITY IN DENERVATED ELECTROPLAX

The glycolytic activity of denervated tissue of Main organ, checked by DR. F. C. G. HosI{IN was decreased by 40-60 % 91-97 days after denervation, whereas the activity of the tissue of Sachs organ after 2 and 25 days was little affected.

lO0~ s0

60~ 40

i2o .£ az U

02 6 I0 i

i

25

i

J

Time a f t e r d e n e r v a t i o n ( d a y s )

Fig. 5. Decrease in choline acetylase a c t i v i t y of tissue of Sachs organ following d e n e r v a t i o n .

TABLE

i

E S T E R A S E A C T I V I T Y OF NORMAL AND 7 8 DAYS DI~NERVATED SACHS ORGAN

Results

are expressed

as/*moles

Condition

Normal

I)enervated

acetylcholine

hydrolyzed

Acetylcholine (molesJ

Activity

per gram

per hour.

•

o.o25 0.005 o.oor

845; 775

o.o25 o.oo5

835; 8oo 314; 316

o.ool

312; 254

375; 3 l o 357; 23o

DISCUSSION

The main aim of the present study was to establish a relationship between the level of choline acetylase activity and the function of the electroplax after denervation measured by the electrical response of an isolated single electroplax. The response to neural stimulation which involves synaptic transmission fails very early, after about 2-3 days. At that time the response to direct stimulation is still hardly affected. Choline acetylase is nearly unchanged, 2 days following denervation. Even after IO days when the response to direct stimulation appeared to be normal, choline acetylase was still high, about 4o % of the initial value. 25 days following denervation when the direct Biochim. Biophys. Acta, 82 (i964) 266 275

274

P. ROSENBERG et al.

response was usually small, graded and non-conducted although sometimes an apparently normal, full-sized conducted direct response was obtained, the choline acetylase had fallen to about I o - I 5 % of the initial value. Similar variations in electrical response were observed at still longer periods of time after denervation (97 days). At that period, choline acetylase activity is indetectable in the Sachs organ and very low in the Main organ, about 2 /xg/g/h. However, the evaluation of the relationship between function and enzyme activity encounters an obstacle similar to that recently described in full detail for the relationship between conduction and acetylcholinesterase activity in axons exposed to D F P (see ref. 29). While the electrical properties have been recorded on individual cells, the choline acetylase activity has been measured in tissue samples weighing o.5 g and containing about 2o cells. This is the minimum amount of tissue for obtaining reliable values of Sachs organ activity with the method used. The value of enzyme activity obtained thus represents an average value. The activity in most of the cells m a y be very low (near o) although perhaps just adequate to maintain a small local non-conducting response consistent with the electrical picture obtained. In a few other cells of the 2o used, the activity may still be lO-15 % of the initial value and adequate for a conducted spike. In the incubation mixture, however, choline acetylase activity might not be detected because the final average value of synthesis would be below the limits of the method used even though several cells still had considerable activity. The electrical activity of individual cells of the Main organ has not been checked since at present no adequate techniques are available for such tests; choline acetylase activity in the Main organ 97 days following denervation was low. Thus it was not possible in the present study to correlate in a quantitative way electrical activity with a minimum of choline acetylase activity. As can be seen from Fig. 5 the choline acetylase activity declines much more slowly than was observed in degenerating peripheral nerve or denervated ganglia 1-a,8. This m a y indicate that only part of the activity is associated with the nerve fibers, including the nerve endings while the rest of the activitv is associated with the electroplax. It is interesting that there is little if any change in cholinesterase activity of denervated Sachs organ, using I, 5 or 25" IO a M acetylcholine as substrate. At the two lower concentrations mainly acetylcholinesterase of the electroplax is assayed whereas at 25" IO-3 M the activity measured is mainly that of the extracellular esterase present in connective tissue which is not acetylcholinesterasO °. It appears that neither of the two enzymes decreased significantly. The values obtained for normal tissue (Table I) are in good agreement with earlier results 3a, estimated on the basis of the average weight of an electroplax. Our finding that acetyleholinesterase is much more resistant than choline acetylase to changes following denervation agrees with earlier findings that following denervation or degeneration acetylcholinesterase does not fall below about 4 0 % of normal4,6, 9-1~, whereas choline acetylase declines to undetectable amounts*-a,8. Only a small fraction, about 5 % of the total cholinesterase activity of the Sachs cell appears to be in the region of the synapsesaL If only the cholinesterase of the nerve endings and synaptic regions declines following denervation, while the activity of the rest of the cell remains unaltered, the decrease in enzyme activity would be small and might easily have been missed. Biochim. Biophys. Acta, 82 (1964) 266-275

ENZYMIC ACTIVITY IN DENERVATED ELECTROPLAX

275

The data described offer some new information about the enzymes forming and splitting acetylcholine in denervated electric tissue. They demonstrate again the difficulties encountered in attempting to correlate changes in enzyme concentration and changes in function of multicellular preparation.

ACKNOWLEDGEMENTS

We thank Dr. D. NACHMANSOHNfor the helpful advice and stimulating discussions during this investigation. We also thank Dr. F. C. G. HOSKIN, Miss E. BARTEL8 and Mr. T. R. PODLESKI for allowing us to report some of their data, and Dr. H. CLAIRE LAWLER for advice concerning the preparation of phosphotransacetylase. This work was supported by the Division of Research Grants and Fellowships, U.S. Public Health Service, Grants No. NB-o33o4 and 2B-5216; by a Special Trainee Fellowship Grant No. BT-579, National Institutes of Health, to P.R.; and by Special Fellowship Grant No. BT-6o6 and Neuropathology Training Grant No. 2B-5o62 from NINDB, U.S. Public Health Service, to E . A . M . H . B . H . is a Special Trainee Fellow of National Institutes of Health, Grant No. BT-526.

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