EXPERIMENTAL
NEUROLOGY
Synthesis
TOMA; Institute and
56,
281--k?!%
(1977)
of Neuromuscular and Denervated KIAUTA,
MIRO
BRZIN
of Pathophysiology, University Defiartment of Pharmacology, Nashville, Received
December 6, 1976;
Cholinesterases Rat Diaphragm AND WOLF-D.
in Innervated
DETTBARN
of Ljubljana, 61105 Ljubljana, Vanderbilt University School of Tennessee 37232 revision
received
February
l Yugoslavia
Medicine,
28, 1977
Quantitative measurements of cholinesterase activity using either acetylcholine (3 mM) in combination with tetramonoisopropylpyrophosphortetramide (10 PM) or butyrylcholine (3 mM) as substrates have been made in junctional and extrajunctional regions of innervated and denervated rat diaphragm preparations. Acetylcholinesterase activity was highly concentrated in the junctional region and a significant portion was present in the extrajunctional region of the niuscle fibers. Butyrylcholinesterase was found evenly distributed in junctional and extrajunctional regions. A significant decrease in the specific aotivities of acetyl- and butyrylcholinesterase was observed in the junctional and extrajunctional regions 3 days after denervation. After in viva irreversible inhibition of cholinesterase activity with phospholine (0.2 mg/kg subcutaneously), and organophosphorus compound, acetyl- and butyrylcholinesterase activities were nearly restored in innervated muscle within 7 days after the initial inhibition. In denervated muscle both enzymes recovered to the activities seen in denervated control muscle. The ,results are discussed in terms of intrinsic muscular control of a part of the junotional cholinesterase activity and the possibility of transsynaptic transport of acetylcholinesterase from the nerve terminal to the postsynaptic membrane.
INTRODUCTION There is considerable evidence supporting the suggestion that nerves exercise control over constituents of the neuromuscular junction as well Abbreviations : ChE-cholinesterases, AChE-acetylcholinesterase, cholinesterase, iso-OMPA-tetramonoisopropylpyrophosphortetramide.
BuChE-butyryI-
1 The authors wish to thank Mr. Vasilij Loboda for excellent technical assistance. This study was supported by Boris Kidrii: Foundation, Ljubljana, by National Institutes of Health Grants 02-008-1, Z-ZF-6 (awarded to T. K. and M. B.) ; ES-00619 and NS,l2348-01 and a research grant-in-aid from the Muscular Dystro,phy Association of America, Inc. (to W-D. D.). Drs. Kiauta and Brzin are at the University of Ljubljana and Dr. Dettbarn is at Vanderbilt University. Send reprint requests to Dr. Dettbarn. 281 Copyright Q 1977 by AcademicPress,Inc. All rights of reproduction in any form reserved.
ISSN 0014-4886
282
KIAUTA,
BRZIN
AND
DETTBARN
as the whole muscle surface membrane (6, 9, 10). Rat skeletal muscle shows a decrease in junctional cholinesterase (ChE) activity within 3 days of denervation which persists at this level without reinnervation for a prolonged period (8). Several hypotheses concerning the role of nerve in controlling muscle have been proposed (6, 7). One possibility is that the nerve supplies one or several trophic factors which control ChE activity. A second is that the nerve merely provides for muscle activity and that excitation controls muscular ChE activity. Neurotrophic factors are supposedly involved in the control of muscle metabolism (6). It has been demonstrated that some postdenervation changes in the muscle (e.g., decrease in resting membrane potential, spread of extrajunctional sensitivity to acetylcholine, and changes in frequency and subsequent disappearance of miniature end-plate potentials) occur sooner if the nerve is transected near the muscle than if it is transected farther away from the muscle (1). Because with lesions distant from or close to the muscle identical effects are produced on muscular activity, the presence of the time differential could best be explained by the presence of a trophic factor. This, however, does not appear to be valid for the control of ChE activity in end-plate and muscle, because the onset of ChE activity decrease after denervation is independent of the length of the remaining nerve stump (8, 16). Experiments on the synthesis of ChE either after irreversible inhibition in innervated and denervated muscle (2, 18, 20) or after muscle fiber destruction (14) provide insight into the mechanisms by which the nerve controls the ChE activity of muscle. Previous experiments studying the synthesis of ChE after irreversible inhibition in innervated and denervated muscle fibers demonstrated either no synthesis (2, 18) or full synthesis (20) of enzyme in denervated muscle. Spontaneous reactivation of phosphorylated ChE is ruled out in those experiments because it has been shown that phospholine and other inhibitors irreversibly inhibit acetylcholinesterase ( AChE) and butyrylcholinesterase ( BuChE) of rat diaphragm, and prolonged washing does not restore enzyme activity (21). Therefore, any recovery of ChE activity after organophosphate inhibition is most probably due to synthesis ob these enzymes. In the present series of experiments, AChE and BuChE activity in junctional and extrajunctional regions of muscle and their de novo synthesis have been studied in normal and denervated rat diaphragm after irreversible inhibition of AChE and BuChE activity. MATERIALS
AND
METHODS
Male Wistar strain albino rats weighing between 100 and 160 g were used, In ‘25 rats, the left hemidiaphragm was denervated by transection of
SYNTHESIS
OF
MUSCLE
CHOLINESTERASES
283
the phrenic nerve about 0.5 cm proximal to the muscle. Of these, 13 denervated control rats were killed 3, 7, or 14 days after denervation. The remaining 12 rats were injected with phospholine [ 0,0-diethyl-S(2-trimethylethylammonium) phosphorothioate methiodide ; Ayerst Pharmaceutical Company, New York, 0.2 mg/kg, subcutaneous] 3 days after denervation and killed 1 h, 7 days, or 14 days after injection. This dose of phospholine causes salivation, diarrhea, body tremor, and muscle twitching. All animals survived without atropine treatment. The diaphragms of four nondenervated rats were used as nondenervated controls. Preparation of Tissue. Animals were anesthesized with ether and the inferior vena cava was ligated under the diaphragm. A plastic cannula was introduced through the right atrium into the intrathoracic part of the vena cava and the diaphragm was perfused with 20 ml Ringer’s solution. The Ringer’s solution used throughout these experiments contained 138 mmol NaCl, 4 mmol KCI, and 1 mmol MgSOl per liter of 50 mM sodium phosphate buffer, pH 7.4. The muscle, with ribs attached, was removed and pinned out in a Petri dish and cleaned of excess connective tissue. Under a dissecting microscope with illumination from below, 3-mm wide strips of the junctional regions of both hemidiaphragms were cut out. Similar strips were cut out from the extrajunctional regions in the vicinity of the ribs, with care to avoid the musculotendineous junction. The muscle samples were weighed (6 to 15 mg, fresh weight) and homogenized in Ringer’s solution with small glass homogenizers. Cholinesterase Assay. AChE activity was determined radiometrically by preincubating the homogenates 1 h at about 0°C with 10 pM tetramonoisopropylpyrophosphortetramide (iso-OMPA) and incubating in a shaking bath 1 h at 38°C with 3 mM acetylcholine iodide and 10 PM iso-OMPA with Ringer’s solution. BuChE activity was determined with 3 mna butyrylcholine iodide as substrate; no inhibitors were used. [ 1-14C] Acetylcholine chloride (The Radiochemical Centre, Amersham, England ; specific activity 11.8 mCi/mmol) and [ 1-“Cl butyrylcholine iodide (New England Nuclear; specific activity 1 to 5 mCi/mmol) were added to the AChE and BuChE incubation media, respectively, to give a final radioactivity of about 0.4 &i/ml. After incubation, lo-p1 aliquots of the incubation mixtures were chromatographed (11). All measurements were done in triplicate. Radioactivity was measured by liquid scintillation spectrometry (Isocap/ 300, Nuclear Chicago) using a previously described liquid scintillation mixture (5). Proteins were determined according to the method of Lowry et al. (12) with bovine serum albumin as standard. The enzyme activity was expressed as specific activity, i.e., activity per milligram protein.
284
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RESULTS Efect of Denervation of AChE and BuChE Activity in Junctional and En-trajunctional Regions of the Diaphragm Mzmle. In the innervated diaphragm, specific junctional AChE activity was about three times higher than the specific activity in the extrajunctional region. Three days after denervation, AChE activity was significantly reduced to approximately 30% in both regions. No recovery of enzyme activity was observed within 14 days of denervation (Table 1). The specific activity of BuChE was only 2.2% of that of AChE in the junctional region, and 62% of that of AChE in the extrajunctional region. Denervation reduced BuChE activity to about 18% of nondenervated control activity in both regions. Within 14 days, however, BuChE activity recovered to between 40% and 50% of the nondenervated control activity (Table 1) . There were no significant differences in the activities of these two enzymes when measured in the innervated left or right hemidiaphragm. Eflects of Denervation on the Recovery of Irreversibly Inhibited AChE and BuChE. The phospholine injection was given on the third day after denervation, because no further significant reduction of enzyme activity was seen after the first 3 days of denervation. One hour after injection, AChE activity in the denervated hemidiaphragm was reduced to 4% and 8% in the junctional and extrajunctional regions, respectively, and in the TABLE
1
Effect of Denervation on Acetylcholinesterase (AChE) and Butyrylcholinesterase (BuChE) Activity in Junctional and Extrajunctional Regions of the Rat Diaphragm Days after denervation
Nondenervated control (N = 4) 3 (iv = 5) 7 (N 14 (N
= 4) = 4)
AChE
activity
Junctional region
Extrajunctional region
35.4 f 6.8a.b (100) 10.8 f 1.6 (30) 9.4 f 1.5
11.6 f 1.8b (100) 4.0 f 0.7 (34) 3.4 f 0.8
(26)
(29) 3.4 f 0.8 (29)
10.4 f 2.0 (29)
BuChE Junctional region
7.8 f 3.3b (100) 1.4 f 0.6
(18) 3.4 f 0.3b (43) 3.2 f 0.6b (41)
activity Extrajunctional region 7.2 f
1.4b
(100) 1.4 f 0.5 (19) 2.6 f O.Sc (36) 3.6 f 1.9 (50)
D Values are X 10m5 moles substrate hydrolyzed/g protein/h (mean f SD) ; percentage of control given in parentheses. b Values significantly different from d-day denervated muscles (P < 0.01). c Value significantly different from d-day denervated muscle (P < 0.05).
SYNTHESIS
OF
MUSCLE
285
CHOLINESTERASES
TABLE
2
Synthesis of Acetylcholinesterase in Denervated and Innervated Hemidiaphragm after Irreversible Inhibition Time after phospholine injection
Denervated hemidiaphragm Junctional region
Nondenervated control (N = 4) lh (iv = 4) 7 days (h = 4) 14 days (h = 4)
Extrajunctional region
35.4 f 6.8”~~ 11.6 f l.gb (100) (100) 1.6 f 0.7 1.0 f 0.4 (4) (8) 9.0 f 3.06 2.6 f 0.6”
(25) 9.4 f
1.96
(26)
(2-4 3.8 f 1.3” (32)
Innervated
hemidiaphragm
Junctional region
Extrajunctional region
40.4 f 6.4b (100) 5.0 l 1.1
12.8 f 2.6b (100) 2.8 zk 0.7
(12) 28.6 f 4.6b (70) 24.8 f 8.1b
(61)
(22) 15.6 f 4.6b
(121)
12.4 f l.Ob (99)
a Values are X 10-s moles substrate hydrolyzed/g protein/h (mean i SD) ; percentage of control given in parentheses. b Values significantly different from the muscles 1 h after phospholine injection (P < 0.01).
innervated hemidiaphragm the reduction was to 12% and 22% in the junctional and extrajunctional regions, respectively. After 1 week, the previously inhibited AChE activity was practically fully restored in the innervated muscle. In the denervated hemidiaphragm a substantially smaller recovery was seen (Table 2) which, nevertheless, was sufficient to almost
s
IOO- I ir
7 dbys TIME AFTER
PHOSPHOLINE
14 days INJECTION
FIG. 1. S,pecific cholinesterase activities in the denervated hemidiaphragm after phospholine inhibition versus denervated control hemidiaph,ragms. @-Junctional AChE, m-junctional BuChE, &-extrajunctional AChE, q -extrajunctional BuChE,
286
KIAUTA,
BRZIN
AND
TABLE Synthesis
of Butyrylcholinesterase Hemidiaphram after
Time after phospholine injection
Nondenervated control (N lh (N = 4) 7 days (N = 4) 14 days (N = 4)
Denervated
= 4)
DETTBARN
3
in Denervated and Innervated Irreversible Inhibition
hemidiaphragm
Innervated Junctional region
hemidiaphragm Extrajunctional region
Junctional region
Extrajunctional region
7.8 f 3.3a*b (100) 0.6 f 0.1 (7) 2.4 f 0.4” (30) 3.6 f 0.9b (46)
7.2 f 1.4” (100) 0.8 f 0.2 (11) 2.0 f 0.6b
7.8 f O.gb (100) 1.0 f 0.2 (13) 2.8 f 0.2b
(19) 4.6 f 0.7b
(27) 3.0 f 0.73 (41)
(36) 3.6 f 0.9b
5.0
(46)
7.4 f
l..sJ
(103)
1.4 f
0.7
(62) f
1.0”
(67)
(1 Values are X 10-s moles substrate hydrolyzed/g protein/h (mean f SD) ; percentage of control given in parentheses. bValues significantly different from the muscles 1 h after phospholine injection (P < 0.01).
fully restore the enzyme activity to the level seen in nontreated, denervated muscle (Fig. 1). BuChE activity also decreased after the phospholine injection, but 14 days later it recovered to 46% and 41% in the junctional and extrajunctional regions of the denervated hemidiaphragm, respectively, and only to 46% and 67% in the respective regions of the innervated hemidiaphragm, compared to the nondenervated control (Table 3). When compared to the denervated control, it is seen that the recovery in the denervated hemidiaphragm was practically complete (Fig. 1). DISCUSSION There have been several reports concerning the recovery of ChE after enzyme inhibition in denervated muscle. After irreversible inhibition of ChE with diisopropylphosphofluoridate, no recovery of enzyme activity was demonstrated in the junctional region of denervated guinea pig muscle (2) or in whole denervated rat muscle ( 18). Other investigators reported practically complete recovery of enzyme activity in denervated rat muscle (20). The conflicting results of these studies can probably be best explained on the basis of different techniques used: histochemical techniques which are not reliable for a quantitative estimation of enzyme activity (2, 20) and the use of muscle homogenates without differentiating between AChE and
SYNTHESIS
OF
MUSCLE
CHOLINESTERASES
287
BuChE activity and between the junctional and extrajunctional regions of the muscle and the neglect of the denervation-induced loss of enzyme ( 18). AS shown by our denervation experiments, AChE and BuChE activities of the junctional, as well as of the extrajunctional, regions of the muscle are, to a large extent, dependent on innervation. However, the fraction of enzyme activity found in the muscle 3 days after denervation is clearly nerveindependent because, as shown by the phospholine experiments, this fraction of AChE and BuChE is synthesized by the muscle under its own intrinsic control. The apparent lack of full recovery in the specific activity of BuChE in the innervated hemidiaphragm may be due to an increase in nonenzyme protein synthesis in the muscle. The AChE activity persisting after denervation (about 300/o of the nondenervated control activity) represents the muscle component of the junctional AChE activity which is nerve-independent. The enzyme activity lost after denervation (about 70% of the nondenervated control activity) represents the nerve-dependent AChE. Despite great experimental efforts, the mechanisms by which the nerve controls this fraction of AChE activity remain unclear. One extensively discussed possibility is the transsynaptic transfer of trophic factors responsible for the control of postsynaptic AChE activity (6). However, the evidence thus far has not been convincing. An alternative possibility is the transsynaptic transfer of AChE itself. The transport of AChE from the perikaryon toward the motor nerve terminal has been confirmed repeatedly, and it is now clear that part of this enzyme is transported at a fast rate (13, 17) and appears to be connected with intraaxonal tubules, whereas the slowly transported AChE is probably bound to the axolemma. Although no definite role for AChE is known in axonal conduction, the presence of AChE in the nerve suggests that the enzyme is being transported to replace the functional AChE involved in neuromuscular transmission. The sites at which the enzyme can be localized, i.e., the presynaptic nerve terminal, the synaptic cleft, and the postsynaptic membrane (3, 19), are not incompatible with this suggestion. Although there is considerable direct evidence for transsynaptic transfer of macromolecules (4, 15)) no direct evidence for the presynaptic secretion of AChE is available as yet. REFERENCES S. S., E. X. ALBUQUERQUE, AND L. GUTH. 1976. Neurotraphic ,regulation of prejunctional and postjunctional membrane at the mammalian motor endplate. Exp. Neurol. 53 : 151-165. 2. FILOGAMO, G., AND G. GABELLA. 1966. Cholinesterase behaviour in the denervated and reinnervated muscles. Acta Anat. 63 : 19!%214. 3. FRIEDENBERG, R. M., AND A. M. SELIGMAN. 1972. Acetylcholinesterase at the myoneural junction : Cytochemical ultrastructure and some biochemical considerations. 1. Histochem. Cytochem. 20 : 771-792. 1. DESHPANDE,
288
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AND
DETTBARN
B. 1971. Transneuronal transfer of radioactivity in the central nervous Science 172 : 177-179. 5. GRUBIC, Z., T. KIAUTA, AND M. BRZIN. 1976. A radiometric method for the determination of choline acetylase activity based on thin-layer chromatography.
4. GRAFSTEIN, system.
Anal. Biocluwz. 74 : 354-358. 6. GUTH, L. 1968. “Trophic” influences of nerve on muscle. Plrysiol. Rev. 48: 645 687. 7. GUTH, L. 1969. “Trophic” effects of vertebrate neurons. Nc~rosci. Rcs. Prog. Bull. 7: l-73. 8. GUTH, L., W. C. BROWN, AND P. K. WATSON. 1967. Studies on the role of nerve impulses and acetylcholine release in the regulation of the cholinesterase activity of muscle. Es-p. Neural. 18 : 443-452. 9. GUTMANN, E. 1976. Neurotrophic relations. AWL Rev. Physiol. 38: 177-216. 10. HARRIS, A. J. 1974. Inductive functions of the nervous system. Amt. Rev. Plzysiol. 36:251-305. 11. LEWIS, M. K., AND M. E. ELDEFRAWI. 1974. A simple, rapid, and quantitative radiometric assay of acetylcholinesterase. Anal. Bioclzem 57 : 588-592. 12. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, AND R. J. RANDALL. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chenz. 193: 265-275. 13. LUBI~SKA, L., AND S. NIEMIERKO. 1971. Velocity and intensity of bidirectional migration of acetylcholinesterase in transected nerves. Brain Rcs. 27: 329-342. 14. MAX, S. R., AND E. X. ALBUQUERQUE. 1975. Neurotrophic regulation of acetylcholinesterase in regenerating skeletal muscle. Exp. Ncwol. 49 : 852-857. 15. NEALE, J. H., E. A. NEALE, AND B. W. AGRANOFF. 1972. Radioautography of the optic tectum of the goldfish after intraocular injection of [“Hlproline. Science 176:407-410. 16. RANISH, N., AND W-D. DETTBARN. 1976. Trophic control of muscle cholinesterase. Neurosci. Abst. 2 : 1036. 17. RANISH, N., AND S. OCHS. 1972. Fast axoplasmic transport of acetylcholinesterase in mammalian nerve fibers. J. Nrzkroc~kcm. 19 : 2641-2649. 18. ROSE, S., AND P. H. GLOW. 1967. Denervation effects on the presumed de sovo synthesis of muscle cholinesterase and the effects of acetylcholine availability on retinal cholinesterase. Exp. NewoZ. 18 : 267-275. 19. SALPETER, M. M., H. PLATTNER, AND A. W. ROGERS. 1972. Quantitative assay of esterases in end plates of mouse diaphragm by elect.ron microscope autoradiography. J. Histocke?tk. Cytochon. 20 : 1059-1068. 20. SONESSON, B., AND S. THESLEFF. 1968. Cholinesterase activity after DFP application in botulinum poisoned, surgically denervated or normally innervated rat skeletal muscles. Life Sci. 7 : 411-417. 21. WELSCH, F., AND W-D. DETTBARN. 1972. Inhibition of cholinesterases of rat diaphragm muscle by o’rganophosphates and spontaneous recovery of enzyme activity in vitro. Biochewa. Pharmacol. 21 : 1039-1049.
NEUROLOGY
Synthesis
TOMA; Institute and
56,
281--k?!%
(1977)
of Neuromuscular and Denervated KIAUTA,
MIRO
BRZIN
of Pathophysiology, University Defiartment of Pharmacology, Nashville, Received
December 6, 1976;
Cholinesterases Rat Diaphragm AND WOLF-D.
in Innervated
DETTBARN
of Ljubljana, 61105 Ljubljana, Vanderbilt University School of Tennessee 37232 revision
received
February
l Yugoslavia
Medicine,
28, 1977
Quantitative measurements of cholinesterase activity using either acetylcholine (3 mM) in combination with tetramonoisopropylpyrophosphortetramide (10 PM) or butyrylcholine (3 mM) as substrates have been made in junctional and extrajunctional regions of innervated and denervated rat diaphragm preparations. Acetylcholinesterase activity was highly concentrated in the junctional region and a significant portion was present in the extrajunctional region of the niuscle fibers. Butyrylcholinesterase was found evenly distributed in junctional and extrajunctional regions. A significant decrease in the specific aotivities of acetyl- and butyrylcholinesterase was observed in the junctional and extrajunctional regions 3 days after denervation. After in viva irreversible inhibition of cholinesterase activity with phospholine (0.2 mg/kg subcutaneously), and organophosphorus compound, acetyl- and butyrylcholinesterase activities were nearly restored in innervated muscle within 7 days after the initial inhibition. In denervated muscle both enzymes recovered to the activities seen in denervated control muscle. The ,results are discussed in terms of intrinsic muscular control of a part of the junotional cholinesterase activity and the possibility of transsynaptic transport of acetylcholinesterase from the nerve terminal to the postsynaptic membrane.
INTRODUCTION There is considerable evidence supporting the suggestion that nerves exercise control over constituents of the neuromuscular junction as well Abbreviations : ChE-cholinesterases, AChE-acetylcholinesterase, cholinesterase, iso-OMPA-tetramonoisopropylpyrophosphortetramide.
BuChE-butyryI-
1 The authors wish to thank Mr. Vasilij Loboda for excellent technical assistance. This study was supported by Boris Kidrii: Foundation, Ljubljana, by National Institutes of Health Grants 02-008-1, Z-ZF-6 (awarded to T. K. and M. B.) ; ES-00619 and NS,l2348-01 and a research grant-in-aid from the Muscular Dystro,phy Association of America, Inc. (to W-D. D.). Drs. Kiauta and Brzin are at the University of Ljubljana and Dr. Dettbarn is at Vanderbilt University. Send reprint requests to Dr. Dettbarn. 281 Copyright Q 1977 by AcademicPress,Inc. All rights of reproduction in any form reserved.
ISSN 0014-4886
282
KIAUTA,
BRZIN
AND
DETTBARN
as the whole muscle surface membrane (6, 9, 10). Rat skeletal muscle shows a decrease in junctional cholinesterase (ChE) activity within 3 days of denervation which persists at this level without reinnervation for a prolonged period (8). Several hypotheses concerning the role of nerve in controlling muscle have been proposed (6, 7). One possibility is that the nerve supplies one or several trophic factors which control ChE activity. A second is that the nerve merely provides for muscle activity and that excitation controls muscular ChE activity. Neurotrophic factors are supposedly involved in the control of muscle metabolism (6). It has been demonstrated that some postdenervation changes in the muscle (e.g., decrease in resting membrane potential, spread of extrajunctional sensitivity to acetylcholine, and changes in frequency and subsequent disappearance of miniature end-plate potentials) occur sooner if the nerve is transected near the muscle than if it is transected farther away from the muscle (1). Because with lesions distant from or close to the muscle identical effects are produced on muscular activity, the presence of the time differential could best be explained by the presence of a trophic factor. This, however, does not appear to be valid for the control of ChE activity in end-plate and muscle, because the onset of ChE activity decrease after denervation is independent of the length of the remaining nerve stump (8, 16). Experiments on the synthesis of ChE either after irreversible inhibition in innervated and denervated muscle (2, 18, 20) or after muscle fiber destruction (14) provide insight into the mechanisms by which the nerve controls the ChE activity of muscle. Previous experiments studying the synthesis of ChE after irreversible inhibition in innervated and denervated muscle fibers demonstrated either no synthesis (2, 18) or full synthesis (20) of enzyme in denervated muscle. Spontaneous reactivation of phosphorylated ChE is ruled out in those experiments because it has been shown that phospholine and other inhibitors irreversibly inhibit acetylcholinesterase ( AChE) and butyrylcholinesterase ( BuChE) of rat diaphragm, and prolonged washing does not restore enzyme activity (21). Therefore, any recovery of ChE activity after organophosphate inhibition is most probably due to synthesis ob these enzymes. In the present series of experiments, AChE and BuChE activity in junctional and extrajunctional regions of muscle and their de novo synthesis have been studied in normal and denervated rat diaphragm after irreversible inhibition of AChE and BuChE activity. MATERIALS
AND
METHODS
Male Wistar strain albino rats weighing between 100 and 160 g were used, In ‘25 rats, the left hemidiaphragm was denervated by transection of
SYNTHESIS
OF
MUSCLE
CHOLINESTERASES
283
the phrenic nerve about 0.5 cm proximal to the muscle. Of these, 13 denervated control rats were killed 3, 7, or 14 days after denervation. The remaining 12 rats were injected with phospholine [ 0,0-diethyl-S(2-trimethylethylammonium) phosphorothioate methiodide ; Ayerst Pharmaceutical Company, New York, 0.2 mg/kg, subcutaneous] 3 days after denervation and killed 1 h, 7 days, or 14 days after injection. This dose of phospholine causes salivation, diarrhea, body tremor, and muscle twitching. All animals survived without atropine treatment. The diaphragms of four nondenervated rats were used as nondenervated controls. Preparation of Tissue. Animals were anesthesized with ether and the inferior vena cava was ligated under the diaphragm. A plastic cannula was introduced through the right atrium into the intrathoracic part of the vena cava and the diaphragm was perfused with 20 ml Ringer’s solution. The Ringer’s solution used throughout these experiments contained 138 mmol NaCl, 4 mmol KCI, and 1 mmol MgSOl per liter of 50 mM sodium phosphate buffer, pH 7.4. The muscle, with ribs attached, was removed and pinned out in a Petri dish and cleaned of excess connective tissue. Under a dissecting microscope with illumination from below, 3-mm wide strips of the junctional regions of both hemidiaphragms were cut out. Similar strips were cut out from the extrajunctional regions in the vicinity of the ribs, with care to avoid the musculotendineous junction. The muscle samples were weighed (6 to 15 mg, fresh weight) and homogenized in Ringer’s solution with small glass homogenizers. Cholinesterase Assay. AChE activity was determined radiometrically by preincubating the homogenates 1 h at about 0°C with 10 pM tetramonoisopropylpyrophosphortetramide (iso-OMPA) and incubating in a shaking bath 1 h at 38°C with 3 mM acetylcholine iodide and 10 PM iso-OMPA with Ringer’s solution. BuChE activity was determined with 3 mna butyrylcholine iodide as substrate; no inhibitors were used. [ 1-14C] Acetylcholine chloride (The Radiochemical Centre, Amersham, England ; specific activity 11.8 mCi/mmol) and [ 1-“Cl butyrylcholine iodide (New England Nuclear; specific activity 1 to 5 mCi/mmol) were added to the AChE and BuChE incubation media, respectively, to give a final radioactivity of about 0.4 &i/ml. After incubation, lo-p1 aliquots of the incubation mixtures were chromatographed (11). All measurements were done in triplicate. Radioactivity was measured by liquid scintillation spectrometry (Isocap/ 300, Nuclear Chicago) using a previously described liquid scintillation mixture (5). Proteins were determined according to the method of Lowry et al. (12) with bovine serum albumin as standard. The enzyme activity was expressed as specific activity, i.e., activity per milligram protein.
284
KIAUTA,
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RESULTS Efect of Denervation of AChE and BuChE Activity in Junctional and En-trajunctional Regions of the Diaphragm Mzmle. In the innervated diaphragm, specific junctional AChE activity was about three times higher than the specific activity in the extrajunctional region. Three days after denervation, AChE activity was significantly reduced to approximately 30% in both regions. No recovery of enzyme activity was observed within 14 days of denervation (Table 1). The specific activity of BuChE was only 2.2% of that of AChE in the junctional region, and 62% of that of AChE in the extrajunctional region. Denervation reduced BuChE activity to about 18% of nondenervated control activity in both regions. Within 14 days, however, BuChE activity recovered to between 40% and 50% of the nondenervated control activity (Table 1) . There were no significant differences in the activities of these two enzymes when measured in the innervated left or right hemidiaphragm. Eflects of Denervation on the Recovery of Irreversibly Inhibited AChE and BuChE. The phospholine injection was given on the third day after denervation, because no further significant reduction of enzyme activity was seen after the first 3 days of denervation. One hour after injection, AChE activity in the denervated hemidiaphragm was reduced to 4% and 8% in the junctional and extrajunctional regions, respectively, and in the TABLE
1
Effect of Denervation on Acetylcholinesterase (AChE) and Butyrylcholinesterase (BuChE) Activity in Junctional and Extrajunctional Regions of the Rat Diaphragm Days after denervation
Nondenervated control (N = 4) 3 (iv = 5) 7 (N 14 (N
= 4) = 4)
AChE
activity
Junctional region
Extrajunctional region
35.4 f 6.8a.b (100) 10.8 f 1.6 (30) 9.4 f 1.5
11.6 f 1.8b (100) 4.0 f 0.7 (34) 3.4 f 0.8
(26)
(29) 3.4 f 0.8 (29)
10.4 f 2.0 (29)
BuChE Junctional region
7.8 f 3.3b (100) 1.4 f 0.6
(18) 3.4 f 0.3b (43) 3.2 f 0.6b (41)
activity Extrajunctional region 7.2 f
1.4b
(100) 1.4 f 0.5 (19) 2.6 f O.Sc (36) 3.6 f 1.9 (50)
D Values are X 10m5 moles substrate hydrolyzed/g protein/h (mean f SD) ; percentage of control given in parentheses. b Values significantly different from d-day denervated muscles (P < 0.01). c Value significantly different from d-day denervated muscle (P < 0.05).
SYNTHESIS
OF
MUSCLE
285
CHOLINESTERASES
TABLE
2
Synthesis of Acetylcholinesterase in Denervated and Innervated Hemidiaphragm after Irreversible Inhibition Time after phospholine injection
Denervated hemidiaphragm Junctional region
Nondenervated control (N = 4) lh (iv = 4) 7 days (h = 4) 14 days (h = 4)
Extrajunctional region
35.4 f 6.8”~~ 11.6 f l.gb (100) (100) 1.6 f 0.7 1.0 f 0.4 (4) (8) 9.0 f 3.06 2.6 f 0.6”
(25) 9.4 f
1.96
(26)
(2-4 3.8 f 1.3” (32)
Innervated
hemidiaphragm
Junctional region
Extrajunctional region
40.4 f 6.4b (100) 5.0 l 1.1
12.8 f 2.6b (100) 2.8 zk 0.7
(12) 28.6 f 4.6b (70) 24.8 f 8.1b
(61)
(22) 15.6 f 4.6b
(121)
12.4 f l.Ob (99)
a Values are X 10-s moles substrate hydrolyzed/g protein/h (mean i SD) ; percentage of control given in parentheses. b Values significantly different from the muscles 1 h after phospholine injection (P < 0.01).
innervated hemidiaphragm the reduction was to 12% and 22% in the junctional and extrajunctional regions, respectively. After 1 week, the previously inhibited AChE activity was practically fully restored in the innervated muscle. In the denervated hemidiaphragm a substantially smaller recovery was seen (Table 2) which, nevertheless, was sufficient to almost
s
IOO- I ir
7 dbys TIME AFTER
PHOSPHOLINE
14 days INJECTION
FIG. 1. S,pecific cholinesterase activities in the denervated hemidiaphragm after phospholine inhibition versus denervated control hemidiaph,ragms. @-Junctional AChE, m-junctional BuChE, &-extrajunctional AChE, q -extrajunctional BuChE,
286
KIAUTA,
BRZIN
AND
TABLE Synthesis
of Butyrylcholinesterase Hemidiaphram after
Time after phospholine injection
Nondenervated control (N lh (N = 4) 7 days (N = 4) 14 days (N = 4)
Denervated
= 4)
DETTBARN
3
in Denervated and Innervated Irreversible Inhibition
hemidiaphragm
Innervated Junctional region
hemidiaphragm Extrajunctional region
Junctional region
Extrajunctional region
7.8 f 3.3a*b (100) 0.6 f 0.1 (7) 2.4 f 0.4” (30) 3.6 f 0.9b (46)
7.2 f 1.4” (100) 0.8 f 0.2 (11) 2.0 f 0.6b
7.8 f O.gb (100) 1.0 f 0.2 (13) 2.8 f 0.2b
(19) 4.6 f 0.7b
(27) 3.0 f 0.73 (41)
(36) 3.6 f 0.9b
5.0
(46)
7.4 f
l..sJ
(103)
1.4 f
0.7
(62) f
1.0”
(67)
(1 Values are X 10-s moles substrate hydrolyzed/g protein/h (mean f SD) ; percentage of control given in parentheses. bValues significantly different from the muscles 1 h after phospholine injection (P < 0.01).
fully restore the enzyme activity to the level seen in nontreated, denervated muscle (Fig. 1). BuChE activity also decreased after the phospholine injection, but 14 days later it recovered to 46% and 41% in the junctional and extrajunctional regions of the denervated hemidiaphragm, respectively, and only to 46% and 67% in the respective regions of the innervated hemidiaphragm, compared to the nondenervated control (Table 3). When compared to the denervated control, it is seen that the recovery in the denervated hemidiaphragm was practically complete (Fig. 1). DISCUSSION There have been several reports concerning the recovery of ChE after enzyme inhibition in denervated muscle. After irreversible inhibition of ChE with diisopropylphosphofluoridate, no recovery of enzyme activity was demonstrated in the junctional region of denervated guinea pig muscle (2) or in whole denervated rat muscle ( 18). Other investigators reported practically complete recovery of enzyme activity in denervated rat muscle (20). The conflicting results of these studies can probably be best explained on the basis of different techniques used: histochemical techniques which are not reliable for a quantitative estimation of enzyme activity (2, 20) and the use of muscle homogenates without differentiating between AChE and
SYNTHESIS
OF
MUSCLE
CHOLINESTERASES
287
BuChE activity and between the junctional and extrajunctional regions of the muscle and the neglect of the denervation-induced loss of enzyme ( 18). AS shown by our denervation experiments, AChE and BuChE activities of the junctional, as well as of the extrajunctional, regions of the muscle are, to a large extent, dependent on innervation. However, the fraction of enzyme activity found in the muscle 3 days after denervation is clearly nerveindependent because, as shown by the phospholine experiments, this fraction of AChE and BuChE is synthesized by the muscle under its own intrinsic control. The apparent lack of full recovery in the specific activity of BuChE in the innervated hemidiaphragm may be due to an increase in nonenzyme protein synthesis in the muscle. The AChE activity persisting after denervation (about 300/o of the nondenervated control activity) represents the muscle component of the junctional AChE activity which is nerve-independent. The enzyme activity lost after denervation (about 70% of the nondenervated control activity) represents the nerve-dependent AChE. Despite great experimental efforts, the mechanisms by which the nerve controls this fraction of AChE activity remain unclear. One extensively discussed possibility is the transsynaptic transfer of trophic factors responsible for the control of postsynaptic AChE activity (6). However, the evidence thus far has not been convincing. An alternative possibility is the transsynaptic transfer of AChE itself. The transport of AChE from the perikaryon toward the motor nerve terminal has been confirmed repeatedly, and it is now clear that part of this enzyme is transported at a fast rate (13, 17) and appears to be connected with intraaxonal tubules, whereas the slowly transported AChE is probably bound to the axolemma. Although no definite role for AChE is known in axonal conduction, the presence of AChE in the nerve suggests that the enzyme is being transported to replace the functional AChE involved in neuromuscular transmission. The sites at which the enzyme can be localized, i.e., the presynaptic nerve terminal, the synaptic cleft, and the postsynaptic membrane (3, 19), are not incompatible with this suggestion. Although there is considerable direct evidence for transsynaptic transfer of macromolecules (4, 15)) no direct evidence for the presynaptic secretion of AChE is available as yet. REFERENCES S. S., E. X. ALBUQUERQUE, AND L. GUTH. 1976. Neurotraphic ,regulation of prejunctional and postjunctional membrane at the mammalian motor endplate. Exp. Neurol. 53 : 151-165. 2. FILOGAMO, G., AND G. GABELLA. 1966. Cholinesterase behaviour in the denervated and reinnervated muscles. Acta Anat. 63 : 19!%214. 3. FRIEDENBERG, R. M., AND A. M. SELIGMAN. 1972. Acetylcholinesterase at the myoneural junction : Cytochemical ultrastructure and some biochemical considerations. 1. Histochem. Cytochem. 20 : 771-792. 1. DESHPANDE,
288
KIAUTA,
BRZIN
AND
DETTBARN
B. 1971. Transneuronal transfer of radioactivity in the central nervous Science 172 : 177-179. 5. GRUBIC, Z., T. KIAUTA, AND M. BRZIN. 1976. A radiometric method for the determination of choline acetylase activity based on thin-layer chromatography.
4. GRAFSTEIN, system.
Anal. Biocluwz. 74 : 354-358. 6. GUTH, L. 1968. “Trophic” influences of nerve on muscle. Plrysiol. Rev. 48: 645 687. 7. GUTH, L. 1969. “Trophic” effects of vertebrate neurons. Nc~rosci. Rcs. Prog. Bull. 7: l-73. 8. GUTH, L., W. C. BROWN, AND P. K. WATSON. 1967. Studies on the role of nerve impulses and acetylcholine release in the regulation of the cholinesterase activity of muscle. Es-p. Neural. 18 : 443-452. 9. GUTMANN, E. 1976. Neurotrophic relations. AWL Rev. Physiol. 38: 177-216. 10. HARRIS, A. J. 1974. Inductive functions of the nervous system. Amt. Rev. Plzysiol. 36:251-305. 11. LEWIS, M. K., AND M. E. ELDEFRAWI. 1974. A simple, rapid, and quantitative radiometric assay of acetylcholinesterase. Anal. Bioclzem 57 : 588-592. 12. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, AND R. J. RANDALL. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chenz. 193: 265-275. 13. LUBI~SKA, L., AND S. NIEMIERKO. 1971. Velocity and intensity of bidirectional migration of acetylcholinesterase in transected nerves. Brain Rcs. 27: 329-342. 14. MAX, S. R., AND E. X. ALBUQUERQUE. 1975. Neurotrophic regulation of acetylcholinesterase in regenerating skeletal muscle. Exp. Ncwol. 49 : 852-857. 15. NEALE, J. H., E. A. NEALE, AND B. W. AGRANOFF. 1972. Radioautography of the optic tectum of the goldfish after intraocular injection of [“Hlproline. Science 176:407-410. 16. RANISH, N., AND W-D. DETTBARN. 1976. Trophic control of muscle cholinesterase. Neurosci. Abst. 2 : 1036. 17. RANISH, N., AND S. OCHS. 1972. Fast axoplasmic transport of acetylcholinesterase in mammalian nerve fibers. J. Nrzkroc~kcm. 19 : 2641-2649. 18. ROSE, S., AND P. H. GLOW. 1967. Denervation effects on the presumed de sovo synthesis of muscle cholinesterase and the effects of acetylcholine availability on retinal cholinesterase. Exp. NewoZ. 18 : 267-275. 19. SALPETER, M. M., H. PLATTNER, AND A. W. ROGERS. 1972. Quantitative assay of esterases in end plates of mouse diaphragm by elect.ron microscope autoradiography. J. Histocke?tk. Cytochon. 20 : 1059-1068. 20. SONESSON, B., AND S. THESLEFF. 1968. Cholinesterase activity after DFP application in botulinum poisoned, surgically denervated or normally innervated rat skeletal muscles. Life Sci. 7 : 411-417. 21. WELSCH, F., AND W-D. DETTBARN. 1972. Inhibition of cholinesterases of rat diaphragm muscle by o’rganophosphates and spontaneous recovery of enzyme activity in vitro. Biochewa. Pharmacol. 21 : 1039-1049.
