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Denervation effects on the presumed de novo synthesis of muscle cholinesterase and the effects of acetylcholine availability on retinal cholinesterase
EXPERIMENTAL
18, 267-275 (1967)
NEUROLOGY
Denervation Synthesis
de Novo
Effects on the Presumed of Muscle Cholinesterase
Effects
of Acetylcholine Retinal
and
Availability
the on
Cholinesterase S. ROSE
Department
of
Physiology,
University
Melbourne,
of
Parkville,
Victoria,
Australia
AND
P. H. GLOWI Department Received
of Psychology, February
University
24, 1967;
Revision
of Adelaide, Received
South March
Australia 27, 1967
Denervationof skeletalmuscleretardsthe presumed de nova synthesis of cholinesterase after the initial reduction of the enzyme by diisopropylfluorophospbate treatment.
Acetylcholine
continuously
infused
into
normal muscle did not affect the vitreous chamber increased the rate of cholinesterase synthesis in the retina. Hemicholinium infused into the vitreous chamber decreased the rate of cholinesterase synthesis in the retina. These results suggest that the availability of acetylcholine influences the presumed de novo synthesis of its regulating enzyme cholinesterase.
cholinesterase activity. Acetylcholineinfusedinto
Introduction
A distinction can be made between the effect of neuromuscular denervation and of disuse on various muscle proteins. Disuse results in muscle atrophy and a loss of muscle protein, but it has little effect on the cholinesterase (ChE) activity of muscle. Denervation results in atrophy, loss of protein and loss of cholinesterase activity. The decrease in ChE activity being faster than the decreasein muscle protein (2, 7). These results suggest that the nerve is specific for the metabolic regulation of at least one muscleprotein, namely, ChE. The synthesis of cholinesterasehas been investigated by first inhibiting the enzyme with diisopropylfluorophosphate (DFP) . Studies of cholinesterase synthesis using this technique in peripheral nerve (8) and in cholinergic neuron (9) have been reported. DFP was chosen for the muscle cholinesterasestudy in this paper becauseit was demonstrated to be an irreversible 1 We acknowledge Agricultural Institute,
the assistance of Mrs. M. Atkinson of the Biometry University of Adelaide, South Australia.
267
Section,
Waite
268
ROSE
AND
GLOW
inhibitor of cholinesterase.The return of cholinesteraseactivity could then probably be attributed to de ~OZJO synthesis (5, 8, 9). The experiments in this paper were designed to determine the quantitative relationship between the innervation of skeletal muscle and the presumed de nova synthesis of muscle ChE after DFP treatment in the rat. Recently this relationship has been studied using a histochemical method for determining ChE activity. It was shown that ChE activity did not recover in denervated chicken muscle but did recover in normal muscle (3). Additional in vivo experiments were designed to study the relationship between the availability of acetylcholine on the activity and synthesis of ChE in muscle and retina. Experiment
1.
De Novo
Synthesis
of ChE and
Innervation
Procedure. Wistar female adult random bred rats weighing between ZOO-300 g were used. They were given an irreversible inhibitor of ChE (DFP) 1 mg/kg into the left foreleg. Four hours later they were anesthetized with chloral hydrate (400 mg/kg). A midline verticle incision was made in the lower abdomenand the plexus of nerves passingto the right and left thigh were exposed. The innervation to the thigh muscle was determined by electrostimulation and the nerve supplies to two muscles,rectus femoris (A) and vastus lateralis (B) of the right leg were sectioned and 5 mm of nerve was removed. Two muscleson the operated side adductor longus (C) and adductor magnus (D) were chosen as nondenervated but were in part functionally altered becausethe movement of the whole limb was impaired. These muscleswill be referred to as nondenervated musclesfrom the operated limb. The animals were killed in groups of six 1, 3, 6, 9 and 14 days postoperatively. The individual muscleswere removed, dissected, weighed and prepared for quantitative ChE assay by the manometric procedure described elsewhere(5). Results and Comments. The effect of denervation on muscle weight is shown in Fig. 1. Muscle decreasedin weight after denervation but not in the nondenervated musclesfrom the operated limb. The details of the effect of the DFP on ChE activity and its recovery with time for various tissueshas been reported (5). The results of ChE activity expressedas ~1 of COz/g wet weight of muscle per minute in the present experiment is set out in Table 1. The muscle from the left leg (control) and the muscle C and D (operated leg but not denervated) showsthe sameinitial fall in ChE activity 24 hours after DFP administration and the same recovery with time as has been reported previously for muscle and other tissue (5). The recovery of the enzyme was characterized by a negatively accelerated increase in ChE activity with time. Approximately 80% of the normal activity returned in 7 days. In contrast the ChE activity of denervated muscIe showed the same
difference
significant
Least
a Split
plot
analysis
B
for
legs
of variance.
ACTIVITY
between:
73.16 112.50 98.00 164.67 125.50
C 50.16 79.66 76.00 116.33 108.83
D
(pl/g/min)
Nondenervated Control
leg (Experimental)
comparisons
37.00 33.83 47.00 36.16 43.16
muscles of the same leg muscles of different legs average effects of muscles legs at one time legs at different times average differences between muscles at the same time muscles at different times
41.50 43.33 49.25 70.66 68.50
1 3 6 9 14
1. 2. 3. 4. 5. 6. 7. 8.
A
Davs
Right
CHOLINESTERASE
Denervated
MEAN
TABLE
50.45 67.33 67.56 96.95 86.50
33.16 74.33 98.25 143.33 105.67
A
1 DFP IN DENERVATED,
All muscles pooled
AFTER
25.83 61.00 58.25 82.16 77.50
B
DISUSED
AND
C
MUSCLES
1% 24.20 24.89 17.09 30.68 42.35 15.30 34.23 43.48
5% 14.21 14.61 10.04 16.62 22.95 8.29 20.09 25.58
44 .oo 90.66 100.25 118.67 113.17
D
lee (Control)
65.16 128.67 105.75 145.50 128.33
Left
CONTROL
42.04 88.66 90.62 122.42 106.17
AU muscles pooled
ROSE
2
4
GLOW
6 TIME
FIG. 1. Log weight propylfluorophosphate.
AND
6
10
12
. 14
(DAYS)
of muscle, denervated, nondenervated
and normal after diiso-
initial decreasebut no significant increase in activity after 7 days and only a small (muscle A) or no (muscle B) increase after 14 days (Fig. 2). The results show that denervation resulted in either no increase or only a modest increasein muscle ChE activity after DFP administration. Since the recovery of ChE activity in normal tissues after DFP administration has been considered as a de nnvo synthesis (5, 8, 9) of this enzyme it can be argued that there is little de nu~o synthesis in the denervated muscle when compared to normal muscle. These findings would support the view that neuromuscular innervation has a significant influence on muscle ChE synthesis.
CHOLINESTERASE
lQ . .z 3
SYNTHESIS
271
6.5
Y x $ 6.0
\ a. w u’
” !I 8
6.0
ii
n 2 $
4.5
4:-,
++v-’ 1
;
s TIME
FIG. 2. Muscle cholinesterase diisopropylfluorophosphate.
Experiment
2.
The
activity,
Effect
s ( DAYS
denervated,
of Acetylcholine
1;
lb
14
)
nondenervated
on Muscle
and
ChE
normal
after
Activity
Procedure. Thirty-six animals were treated with DFP and the nerves sectioned as in Experiment 1. In addition a polyethylene catheter was inserted into the right iliac artery and 5 to 10 ml/day of 0.6-170 acetylcholine was infused continuously in the unanesthetized and unrestrained rat. The methods for arterial infusion have been described ( 11, 12). The catheter in the iliac artery tended to cause spasm in that vessel, a factor probably responsible for the development of gangrene of the leg and death of the animal within 2 to 3 days. As catheters inserted into the carotid artery do not cause a spasm, continuous arterial infusion of the head and neck was carried out. Five to 10 ml/day of 2 or 3% acetylcholine was continuously infused into the right
272
ROSE
AND
GLOW
carotid artery of normal Wistar nondenervated rats and after 7 days of treatment the right and left masseter muscle were removed and assayed as before. The results shown in Table 2 indicate that acetylcholine infused into the arterial supply of normal muscle did not alter the ChE activity of that muscle. The infusion of acetylcholine into the right carotid artery caused blood-stained tears to flow from the right eye thus indicating that there was a significant increase in the acetylcholine concentration of the blood supply of the head and neck. TABLE NORMAL
MUSCLE
Left
ChEa
AFTER
INTRACAROTID
2 INFUSION
noninfused masseter
ACh
FOR
7
DAYS
4.0 3.3 5.7 3.0 4.7 6.0 6.7 4.7 6.0 4.7 10.0
7.3 5.7 8.0 8.0 7.3 7.7 7.0 4.7
S.0
5.7 6.7 7.7 6.7 7.0 7.0 5.7 t = 0.091;
2%
Right infused masseter
4.7 6.0 4.3 2.3 40 6.0 6.7 5.3 5.3 6.0 9.0
(1 pl COa/g/min.
WITH
DF=18;
P = N.S.
As it has been shown that retinal ChE can be protected by an intravitreous injection of acetylcholine (lo), one interpretation for the muscle results is that there is a barrier between the blood and muscle. This possibility was examined by administering acetylcholine intra-arterially to see whether such infusions protected the nondenervated muscle from phosphorylation by systemically administered DFP. Acetylcholine (3% at the rate of 20 ml/day) was given continuously into the right carotid artery. During the infusion DFP (1 mg/kg) was given systemically. Two hours after the administration of DFP and while the acetylcholine infusion was still in progress,the animals were killed, the right and left masseterremoved and assayedfor ChE activity. Results and Comments.The results shown in Table 3 indicate that acetylcholine given intra-arterially did not protect the muscle ChE from phos-
CHOLINESTERASE
273
SYNTHESIS
phorylation by systemic DFP. These results taken in conjunction with the intravitreous result suggest that there is a blood muscle barrier for acetylcholine. MUSCLE
ChE@
3%
AFTER
TREATED
TABLE ACETYLCH~LINE WITH
3 INTRACZAROTID
DFP
SYSTEMIC
INFUSION
Infused side masseter muscle
Noninfused side masseter muscle
4.7 2.0 2.7 5.3 4.0 4.0
4.0 3.0 2.7 3.3 4.0 3.7
a p1 COz/g/min. Experiment
t
= 0.824; 3.
DF=5; Acetylcholine of the
INTO
RAT
(1 mg/kg)
P = N.S. and
Cholinesterase
Activity
Retina
Procedure. It has been shown that there is a blood retina barrier but no barrier between the vitreous chamber and the retina for acetylcholine. Therefore, experiments were designed to determine the effect of continuous intravitreous infusion of acetylcholine on retinal cholinesterase. Animals were given systematically DFP 1 mg/kg and an opaque plastic contact lens was placed over the right eye. The details of the technique and result of this treatment have been described elsewhere (4). An opaque contact lens retards the rate of presumed de novo synthesis of ChE. In six animals prepared in this fashion, a continuous infusion of 1 ml/day of 3% acetylcholine for 3 days was given into the vitreous chamber. The infusion methodology and technique of inserting the catheter into the vitreous chamber have been reported ( 12). In a further group of six animals which had received DFP (1 mg/kg) systemically, hemicholinium, a drug which inhibits the acetylcholine synthesis (1) was infused continuously into the vitreous chamber (5 mg/ml at the rate of 1 ml/day) for 1 week. Hemicholinium in the doses used is not an anticholinesterase. This was shown by adding hemicholinium to the manometric assay system to make the final concentration of this drug 1O-3 to lo-? molar. This result was confirmed by single injections of 0.02 ml of hemicholinium (5 mg/ml) into the vitreous chamber of one eye (experimental) and the same volume of saline into the other eye (control). ChE activity of the control and experimental retina was of the same magnitude and not significantly different from any two retinas of normal animals. Results and Comments. Acetylcholine infused into the vitreous chamber can affect the slower rate of ChE synthesis after systemic administration of
274
ROSE
AND
GLOW
DF = 10; DFP consequent on wearing an opaque contact lens (t = 2.3 ; P = 0.05). Hemicholinium infused into the vitreous chamber of the eye of DFP-treated animals causes a significant fall in the rate of ChE synthesis P = 0.1) . These results indicate of the retina (t = 2.14; DF=S; that acetylcholine level in the retina influences the retinal ChE activity in a manner suggesting a substrate enzyme relationship. Discussion
It seems reasonably clear that denervation retards the presumed de nova synthesis of ChE after the initial reduction of the enzyme by DFP treatment. Two interpretations of these findings are possible. The one lays emphasis on substrate availability regulating enzyme synthesis as found in other biological systems. This argument is supported by the results of chronic intravitreous infusions of drugs. Infused acetylcholine offsets the retarding influence of light deprivation on the de nova synthesis of ChE. On the other hand hemicholinium decreased the de nova synthesis of ChE. The other interpretation of the muscle denervation findings stressesthe possibility of a trophic influence of innervation by virtue of the transfer of specific substances from the axon (13). Such trophic influence might either regulate ChE synthesis or even constitute a transcellular movement of ChE per se from nerve to muscle. The infusion of exogenousacetylcholine did not influence ChE synthesis in nondenervated muscle and the effect of acetylcholine on denervated muscle was technically unsuccessful.The explanation for the lack of effect of infused acetylcholine could arise from an inability of blood-borne acetylcholine to penetrate into the sites of muscle ChE synthesis. An alternative explanation for these results is suggestedby the effect of acetyl p methylcholine on the cholinesteraselevel of chicken embryo muscle in tissue culture (6). It is presumed that no barrier to the diffusion of substrates exists in tissue culture. Despite this fi methylcholine had only a slight effect on the synthesis of muscle ChE. The results from the in vivo and in vitro experiments suggestthat acetylcholine availability may not be the principal condition for ChE synthesis in muscle. References 1. 2. 3. 4.
BIRKS, R., and F. C. MACINTOSH. 1961. Acetylcholine metabolism of a sympathetic ganglion. Can. J. Biochem. Physiol. 39: 787-827. ECCLES, J. C. 1944. Investigations on muscle atrophies arising from disuse and tenotomy. J. Physiol. London 103: 253-266. FDXAMO, G., and G. GABELLA. 1966. Cholinesterase behaviour in the denervated and reinnervated muscles. Acta Anat. 63: 199-214. Grow, P. H., and S. ROSE. 1966. Activity of cholinesterase in the retina with different levels of physiological stimulation. Australian J. Exptl. Biol. Med. Sci. 44: 65-72.
CHOLINESTERASE
5.
6. 7.
8. 9. 10. 11. 12. 13.
SYNTHESIS
27.5
GLOW, P. H., S. ROSE, and A. RICHARDSON. 1966. The effect of acute and chronic treatment with diisopropyl fluorophosphate on cholinesterase activities of some tissues of the rat. Australian J. Exptl. Biol. Med. Sci. 44: 73-86. GOODWIN, B. C., and 1. W. SIZER. 1965. Effects of spinal cord and substrate on acetylcholinesterase in chick embryonic skeletal muscle. Develop. Biol. 11: 1316-153. GUTH, L., R. W. ALBERS, and W. C. BROWN. 1964. Quantitative changes in cholinesterase activity of denervated muscle fibers and sole plates. Exptl. Neural. 10: 236-250. KOENIG, E., and G. B. KOELLE. 1960. Acetylcholinesterase regeneration in peripheral nerve after irreversible inactivation. Science 182: 1249-1250. KOENIG, E., and G. B. KOELLE. 1961. Mode of regeneration of acetylcholinesterase in choline@ neurons following irreversible inactivation. J. Neurochem. 8: 169-188. ROSE, S., and P. H. GLOW. 1965. Effects of intravitreous injection of drugs on the cholinesterase of the retina. Australian 1. Exptl. Biol. Med. Sci. 142: 737-742. ROSE, S., and P. H. GLOW. 1966. Arterial infusion of acetylcholine and thyroid activity. Natwe 212: 616. ROSE, S., and J. NELSON. 1967. Continuous injection methodology in biology. Gamer Chemotherapy Rept. (in press). WEISS, P. 1960. The concept of perpetual neuronal growth and proximo-distal substance convection, p. 254. In “Fourth Intern. Neurochem. Symposium,” Pergamon Press, Oxford.
18, 267-275 (1967)
NEUROLOGY
Denervation Synthesis
de Novo
Effects on the Presumed of Muscle Cholinesterase
Effects
of Acetylcholine Retinal
and
Availability
the on
Cholinesterase S. ROSE
Department
of
Physiology,
University
Melbourne,
of
Parkville,
Victoria,
Australia
AND
P. H. GLOWI Department Received
of Psychology, February
University
24, 1967;
Revision
of Adelaide, Received
South March
Australia 27, 1967
Denervationof skeletalmuscleretardsthe presumed de nova synthesis of cholinesterase after the initial reduction of the enzyme by diisopropylfluorophospbate treatment.
Acetylcholine
continuously
infused
into
normal muscle did not affect the vitreous chamber increased the rate of cholinesterase synthesis in the retina. Hemicholinium infused into the vitreous chamber decreased the rate of cholinesterase synthesis in the retina. These results suggest that the availability of acetylcholine influences the presumed de novo synthesis of its regulating enzyme cholinesterase.
cholinesterase activity. Acetylcholineinfusedinto
Introduction
A distinction can be made between the effect of neuromuscular denervation and of disuse on various muscle proteins. Disuse results in muscle atrophy and a loss of muscle protein, but it has little effect on the cholinesterase (ChE) activity of muscle. Denervation results in atrophy, loss of protein and loss of cholinesterase activity. The decrease in ChE activity being faster than the decreasein muscle protein (2, 7). These results suggest that the nerve is specific for the metabolic regulation of at least one muscleprotein, namely, ChE. The synthesis of cholinesterasehas been investigated by first inhibiting the enzyme with diisopropylfluorophosphate (DFP) . Studies of cholinesterase synthesis using this technique in peripheral nerve (8) and in cholinergic neuron (9) have been reported. DFP was chosen for the muscle cholinesterasestudy in this paper becauseit was demonstrated to be an irreversible 1 We acknowledge Agricultural Institute,
the assistance of Mrs. M. Atkinson of the Biometry University of Adelaide, South Australia.
267
Section,
Waite
268
ROSE
AND
GLOW
inhibitor of cholinesterase.The return of cholinesteraseactivity could then probably be attributed to de ~OZJO synthesis (5, 8, 9). The experiments in this paper were designed to determine the quantitative relationship between the innervation of skeletal muscle and the presumed de nova synthesis of muscle ChE after DFP treatment in the rat. Recently this relationship has been studied using a histochemical method for determining ChE activity. It was shown that ChE activity did not recover in denervated chicken muscle but did recover in normal muscle (3). Additional in vivo experiments were designed to study the relationship between the availability of acetylcholine on the activity and synthesis of ChE in muscle and retina. Experiment
1.
De Novo
Synthesis
of ChE and
Innervation
Procedure. Wistar female adult random bred rats weighing between ZOO-300 g were used. They were given an irreversible inhibitor of ChE (DFP) 1 mg/kg into the left foreleg. Four hours later they were anesthetized with chloral hydrate (400 mg/kg). A midline verticle incision was made in the lower abdomenand the plexus of nerves passingto the right and left thigh were exposed. The innervation to the thigh muscle was determined by electrostimulation and the nerve supplies to two muscles,rectus femoris (A) and vastus lateralis (B) of the right leg were sectioned and 5 mm of nerve was removed. Two muscleson the operated side adductor longus (C) and adductor magnus (D) were chosen as nondenervated but were in part functionally altered becausethe movement of the whole limb was impaired. These muscleswill be referred to as nondenervated musclesfrom the operated limb. The animals were killed in groups of six 1, 3, 6, 9 and 14 days postoperatively. The individual muscleswere removed, dissected, weighed and prepared for quantitative ChE assay by the manometric procedure described elsewhere(5). Results and Comments. The effect of denervation on muscle weight is shown in Fig. 1. Muscle decreasedin weight after denervation but not in the nondenervated musclesfrom the operated limb. The details of the effect of the DFP on ChE activity and its recovery with time for various tissueshas been reported (5). The results of ChE activity expressedas ~1 of COz/g wet weight of muscle per minute in the present experiment is set out in Table 1. The muscle from the left leg (control) and the muscle C and D (operated leg but not denervated) showsthe sameinitial fall in ChE activity 24 hours after DFP administration and the same recovery with time as has been reported previously for muscle and other tissue (5). The recovery of the enzyme was characterized by a negatively accelerated increase in ChE activity with time. Approximately 80% of the normal activity returned in 7 days. In contrast the ChE activity of denervated muscIe showed the same
difference
significant
Least
a Split
plot
analysis
B
for
legs
of variance.
ACTIVITY
between:
73.16 112.50 98.00 164.67 125.50
C 50.16 79.66 76.00 116.33 108.83
D
(pl/g/min)
Nondenervated Control
leg (Experimental)
comparisons
37.00 33.83 47.00 36.16 43.16
muscles of the same leg muscles of different legs average effects of muscles legs at one time legs at different times average differences between muscles at the same time muscles at different times
41.50 43.33 49.25 70.66 68.50
1 3 6 9 14
1. 2. 3. 4. 5. 6. 7. 8.
A
Davs
Right
CHOLINESTERASE
Denervated
MEAN
TABLE
50.45 67.33 67.56 96.95 86.50
33.16 74.33 98.25 143.33 105.67
A
1 DFP IN DENERVATED,
All muscles pooled
AFTER
25.83 61.00 58.25 82.16 77.50
B
DISUSED
AND
C
MUSCLES
1% 24.20 24.89 17.09 30.68 42.35 15.30 34.23 43.48
5% 14.21 14.61 10.04 16.62 22.95 8.29 20.09 25.58
44 .oo 90.66 100.25 118.67 113.17
D
lee (Control)
65.16 128.67 105.75 145.50 128.33
Left
CONTROL
42.04 88.66 90.62 122.42 106.17
AU muscles pooled
ROSE
2
4
GLOW
6 TIME
FIG. 1. Log weight propylfluorophosphate.
AND
6
10
12
. 14
(DAYS)
of muscle, denervated, nondenervated
and normal after diiso-
initial decreasebut no significant increase in activity after 7 days and only a small (muscle A) or no (muscle B) increase after 14 days (Fig. 2). The results show that denervation resulted in either no increase or only a modest increasein muscle ChE activity after DFP administration. Since the recovery of ChE activity in normal tissues after DFP administration has been considered as a de nnvo synthesis (5, 8, 9) of this enzyme it can be argued that there is little de nu~o synthesis in the denervated muscle when compared to normal muscle. These findings would support the view that neuromuscular innervation has a significant influence on muscle ChE synthesis.
CHOLINESTERASE
lQ . .z 3
SYNTHESIS
271
6.5
Y x $ 6.0
\ a. w u’
” !I 8
6.0
ii
n 2 $
4.5
4:-,
++v-’ 1
;
s TIME
FIG. 2. Muscle cholinesterase diisopropylfluorophosphate.
Experiment
2.
The
activity,
Effect
s ( DAYS
denervated,
of Acetylcholine
1;
lb
14
)
nondenervated
on Muscle
and
ChE
normal
after
Activity
Procedure. Thirty-six animals were treated with DFP and the nerves sectioned as in Experiment 1. In addition a polyethylene catheter was inserted into the right iliac artery and 5 to 10 ml/day of 0.6-170 acetylcholine was infused continuously in the unanesthetized and unrestrained rat. The methods for arterial infusion have been described ( 11, 12). The catheter in the iliac artery tended to cause spasm in that vessel, a factor probably responsible for the development of gangrene of the leg and death of the animal within 2 to 3 days. As catheters inserted into the carotid artery do not cause a spasm, continuous arterial infusion of the head and neck was carried out. Five to 10 ml/day of 2 or 3% acetylcholine was continuously infused into the right
272
ROSE
AND
GLOW
carotid artery of normal Wistar nondenervated rats and after 7 days of treatment the right and left masseter muscle were removed and assayed as before. The results shown in Table 2 indicate that acetylcholine infused into the arterial supply of normal muscle did not alter the ChE activity of that muscle. The infusion of acetylcholine into the right carotid artery caused blood-stained tears to flow from the right eye thus indicating that there was a significant increase in the acetylcholine concentration of the blood supply of the head and neck. TABLE NORMAL
MUSCLE
Left
ChEa
AFTER
INTRACAROTID
2 INFUSION
noninfused masseter
ACh
FOR
7
DAYS
4.0 3.3 5.7 3.0 4.7 6.0 6.7 4.7 6.0 4.7 10.0
7.3 5.7 8.0 8.0 7.3 7.7 7.0 4.7
S.0
5.7 6.7 7.7 6.7 7.0 7.0 5.7 t = 0.091;
2%
Right infused masseter
4.7 6.0 4.3 2.3 40 6.0 6.7 5.3 5.3 6.0 9.0
(1 pl COa/g/min.
WITH
DF=18;
P = N.S.
As it has been shown that retinal ChE can be protected by an intravitreous injection of acetylcholine (lo), one interpretation for the muscle results is that there is a barrier between the blood and muscle. This possibility was examined by administering acetylcholine intra-arterially to see whether such infusions protected the nondenervated muscle from phosphorylation by systemically administered DFP. Acetylcholine (3% at the rate of 20 ml/day) was given continuously into the right carotid artery. During the infusion DFP (1 mg/kg) was given systemically. Two hours after the administration of DFP and while the acetylcholine infusion was still in progress,the animals were killed, the right and left masseterremoved and assayedfor ChE activity. Results and Comments.The results shown in Table 3 indicate that acetylcholine given intra-arterially did not protect the muscle ChE from phos-
CHOLINESTERASE
273
SYNTHESIS
phorylation by systemic DFP. These results taken in conjunction with the intravitreous result suggest that there is a blood muscle barrier for acetylcholine. MUSCLE
ChE@
3%
AFTER
TREATED
TABLE ACETYLCH~LINE WITH
3 INTRACZAROTID
DFP
SYSTEMIC
INFUSION
Infused side masseter muscle
Noninfused side masseter muscle
4.7 2.0 2.7 5.3 4.0 4.0
4.0 3.0 2.7 3.3 4.0 3.7
a p1 COz/g/min. Experiment
t
= 0.824; 3.
DF=5; Acetylcholine of the
INTO
RAT
(1 mg/kg)
P = N.S. and
Cholinesterase
Activity
Retina
Procedure. It has been shown that there is a blood retina barrier but no barrier between the vitreous chamber and the retina for acetylcholine. Therefore, experiments were designed to determine the effect of continuous intravitreous infusion of acetylcholine on retinal cholinesterase. Animals were given systematically DFP 1 mg/kg and an opaque plastic contact lens was placed over the right eye. The details of the technique and result of this treatment have been described elsewhere (4). An opaque contact lens retards the rate of presumed de novo synthesis of ChE. In six animals prepared in this fashion, a continuous infusion of 1 ml/day of 3% acetylcholine for 3 days was given into the vitreous chamber. The infusion methodology and technique of inserting the catheter into the vitreous chamber have been reported ( 12). In a further group of six animals which had received DFP (1 mg/kg) systemically, hemicholinium, a drug which inhibits the acetylcholine synthesis (1) was infused continuously into the vitreous chamber (5 mg/ml at the rate of 1 ml/day) for 1 week. Hemicholinium in the doses used is not an anticholinesterase. This was shown by adding hemicholinium to the manometric assay system to make the final concentration of this drug 1O-3 to lo-? molar. This result was confirmed by single injections of 0.02 ml of hemicholinium (5 mg/ml) into the vitreous chamber of one eye (experimental) and the same volume of saline into the other eye (control). ChE activity of the control and experimental retina was of the same magnitude and not significantly different from any two retinas of normal animals. Results and Comments. Acetylcholine infused into the vitreous chamber can affect the slower rate of ChE synthesis after systemic administration of
274
ROSE
AND
GLOW
DF = 10; DFP consequent on wearing an opaque contact lens (t = 2.3 ; P = 0.05). Hemicholinium infused into the vitreous chamber of the eye of DFP-treated animals causes a significant fall in the rate of ChE synthesis P = 0.1) . These results indicate of the retina (t = 2.14; DF=S; that acetylcholine level in the retina influences the retinal ChE activity in a manner suggesting a substrate enzyme relationship. Discussion
It seems reasonably clear that denervation retards the presumed de nova synthesis of ChE after the initial reduction of the enzyme by DFP treatment. Two interpretations of these findings are possible. The one lays emphasis on substrate availability regulating enzyme synthesis as found in other biological systems. This argument is supported by the results of chronic intravitreous infusions of drugs. Infused acetylcholine offsets the retarding influence of light deprivation on the de nova synthesis of ChE. On the other hand hemicholinium decreased the de nova synthesis of ChE. The other interpretation of the muscle denervation findings stressesthe possibility of a trophic influence of innervation by virtue of the transfer of specific substances from the axon (13). Such trophic influence might either regulate ChE synthesis or even constitute a transcellular movement of ChE per se from nerve to muscle. The infusion of exogenousacetylcholine did not influence ChE synthesis in nondenervated muscle and the effect of acetylcholine on denervated muscle was technically unsuccessful.The explanation for the lack of effect of infused acetylcholine could arise from an inability of blood-borne acetylcholine to penetrate into the sites of muscle ChE synthesis. An alternative explanation for these results is suggestedby the effect of acetyl p methylcholine on the cholinesteraselevel of chicken embryo muscle in tissue culture (6). It is presumed that no barrier to the diffusion of substrates exists in tissue culture. Despite this fi methylcholine had only a slight effect on the synthesis of muscle ChE. The results from the in vivo and in vitro experiments suggestthat acetylcholine availability may not be the principal condition for ChE synthesis in muscle. References 1. 2. 3. 4.
BIRKS, R., and F. C. MACINTOSH. 1961. Acetylcholine metabolism of a sympathetic ganglion. Can. J. Biochem. Physiol. 39: 787-827. ECCLES, J. C. 1944. Investigations on muscle atrophies arising from disuse and tenotomy. J. Physiol. London 103: 253-266. FDXAMO, G., and G. GABELLA. 1966. Cholinesterase behaviour in the denervated and reinnervated muscles. Acta Anat. 63: 199-214. Grow, P. H., and S. ROSE. 1966. Activity of cholinesterase in the retina with different levels of physiological stimulation. Australian J. Exptl. Biol. Med. Sci. 44: 65-72.
CHOLINESTERASE
5.
6. 7.
8. 9. 10. 11. 12. 13.
SYNTHESIS
27.5
GLOW, P. H., S. ROSE, and A. RICHARDSON. 1966. The effect of acute and chronic treatment with diisopropyl fluorophosphate on cholinesterase activities of some tissues of the rat. Australian J. Exptl. Biol. Med. Sci. 44: 73-86. GOODWIN, B. C., and 1. W. SIZER. 1965. Effects of spinal cord and substrate on acetylcholinesterase in chick embryonic skeletal muscle. Develop. Biol. 11: 1316-153. GUTH, L., R. W. ALBERS, and W. C. BROWN. 1964. Quantitative changes in cholinesterase activity of denervated muscle fibers and sole plates. Exptl. Neural. 10: 236-250. KOENIG, E., and G. B. KOELLE. 1960. Acetylcholinesterase regeneration in peripheral nerve after irreversible inactivation. Science 182: 1249-1250. KOENIG, E., and G. B. KOELLE. 1961. Mode of regeneration of acetylcholinesterase in choline@ neurons following irreversible inactivation. J. Neurochem. 8: 169-188. ROSE, S., and P. H. GLOW. 1965. Effects of intravitreous injection of drugs on the cholinesterase of the retina. Australian 1. Exptl. Biol. Med. Sci. 142: 737-742. ROSE, S., and P. H. GLOW. 1966. Arterial infusion of acetylcholine and thyroid activity. Natwe 212: 616. ROSE, S., and J. NELSON. 1967. Continuous injection methodology in biology. Gamer Chemotherapy Rept. (in press). WEISS, P. 1960. The concept of perpetual neuronal growth and proximo-distal substance convection, p. 254. In “Fourth Intern. Neurochem. Symposium,” Pergamon Press, Oxford.