Acta physiol. scand. 1975. 93. 525-530 From the Institute of Physiology, University of Lund, Sweden
Compensatory Increase in Choline Acetyltransferase Activity in Salivary Glands and Diaphragm Muscle of the Rat BY JORGEN
EKSTROM
Received 29 October 1974
Abstract EKSTROM, J. Compensatory increase in choline acetyltransferase activity in salivary glands and diaphragm muscle of the rat. Acta physiol. scand. 1975. 93. 525-530. When the function of salivary glands was abolished by applying ligatures to their ducts and the function of one half of the diaphragm muscle was abolished by sectioning of its phrenic nerve, the choline acetyltransferase activity was found to be increased in not duct-ligated glands and in the intact hemidiaphragm 4 weeks later. The increase was not seen within the first week. The increase in activity appears to be particularly manifested in the nerve endings, since it was seen in the hemidiaphragm but not in the phrenic nerve. Increased stream of impulses in the efferent nerves is thought to be the cause of this increase in choline acetyltransferase activity. Key words: Choline acetyltransfeidse activity, salivary glands, diaphragm muscle
When the preganglionic parasympathetic salivary nerve is cut the ability to form acetylcholine in the postganglionic neurones of the gland decreases, as judged from the reduction of choline acetyltransferase activity (Nordenfelt 1964, Ekstrom and Holmberg 1972 a). A fall in the activity of this enzyme of about the same magnitude can be induced in the parotid gland of the rat by keeping the animal on a liquid diet (Ekstrom 1973). The opposite, i.e. a rise in the enzyme activity occurs in the gland when the rat is fed on a dry, cellulose rich diet or when the animal is atropinized causing dryness of the oral mucosa (Ekstrom 1974). The results of these studies suggest that the choline acetyltransferase activity is depending on the impulse-propagating activity of the neurones, as originally pointed out by Nordenfelt (1964); the enzyme activity declines when the flow of secretory impulses in the neurones is lost or reduced and it increases when the impulse traffic is augmented. Observations on skeletal muscles show that disuse is followed by a decrease in choline acetyltransferase activity (Gutmann, TuEek and HanzlikovB 1969, Snyder, Rifenberick and Max 1973, Diamond, Milfay and Franklin 1974). However, opinions differ as to the effect of overuse. Thus an increased choline acetyltransferase activity was found by Snyder et al. (1973) in the plantaris muscle of the rat, when the tendon of the synergistic muscles, gastroc525
526
JORGEN EKSTROM
nemius and soleus, had been severed for 8 days, while Diamond et al. (1974) were unable to demonstrate any increase in the rat gastrocnemius muscle 7 days after the tendons of the soleus and plantaris muscles had been cut. When saliva is prevented from reaching the oral cavity of the rat by ligation of the ducts of some of the salivary glands the reflexly elicited stimulation of the other not duct-ligated glands appears to be augmented, as judged from glandular enlargement (Wells and Peronace 1964, Elmer and Ohlin 1969) and increased secretion (Elm& and Ohlin 1969). In the present study the choline acetyltransferase activity was estimated in such non-ligated glands. As in salivary glands (see Nordenfelt 1965 a, Ekstrom and Holmberg 1972 b) the acetylcholine forming enzyme is in the diaphragm muscle confined to the cholinergic neurones (Hebb, KrnjeviC and Silver 1964, Emmelin, Nordenfelt and Perec 1966, Israel 1970). In the present investigation the enzyme activity was determined in one half of the muscle and in its phrenic nerve when the nerve on the contralateral side of the rat had been sectioned in advance. This procedure seems to lead to an increase in the stimulation of the innervated hemidiaphragm since, according to the result of preliminary experiments, the respiratory frequency of the rat rose.
Methods 74 male rats of a Sprague-Dawley strain bred at this Institute were used. The rats were at the start of the experiments 3-5 months old and weighed 260-380 g. They were given a standard pelleted diet and water ad libitum. The experiments were performed as follows: Sa/iiwr.v glands. The rats were anaesthetized with ether. In the first series the parotid duct on the left side and the ducts of the submaxillary and the sublingual glands on both sides were tied with a fine silk thread, the ducts of the latter two glands close to hilus causing at the same time an interruption of the parasympathetic nerve supply. In the second series both parotid ducts and those of the submaxillary and the sublingual glands on the left side were ligated. At the end of the experimental period the rats were killed with ether and weighed. The position of the ligatures was controlled and macroscopical changes of the ductligated glands were looked for, such as dark red colour and atrophy. In the first series of experiments the nonligated right parotid glands from 5 rats were examined 3 days after the ligation procedure, glands from another 5 rats 4 days later, and from 9 rats 28 days after the beginning of the investigation. In the second series the submaxillary and the sublingual glands on the right side of 9 rats were studied after 28 days, too. When the glands had been removed, they were cleaned, washed in saline, pressed gently between gauze pads and weighed. Since dry weight is considered more reliable, the weight of the acetone dried powder was used. As control glands served the corresponding ones of litter mates to duct-ligated rats; in two cases the control rat was common to both the series of experiments. Thus 54 rats were used. Phrenic neriv and diaphragm. The rats were anaesthetized with pentobarbitone, 60 mg/kp i.p. At the same time atropine sulphate, 1 mg/kg i.p., was given in order t o suppress bronchial secretion. Artificial respiration was given by a pump through a fine glass tube which was inserted into the trachea through a small cut. Between 2 ribs the right phrenic nerve was sectioned in the upper part of the thorax and a nerve segment of about 5 mm was removed. The intrathoracic approach was chosen, since some of the cervical branches forming the phrenic nerve otherwise might escape the section when done in the neck region (Sola and Martin 1953, Emmelin and Malm 1965). The wounds were sutured and the artificial respiration was stopped. After 28 days the respiratory frequency of the operated rat was estimated, when the animal seemed to be at rest; the frequency was also determined in the litter mate used as control. The rats were then killed with ether and weighed. The right sectioned phrenic nerve was inspected. In some instances signs of beginning nerve regeneration could be observed in the form of anatomical connection with the proximal part of the phrenic nerve or intercostal nerves. The left phrenic nerve was cut close to the diaphragm and taken out. A piece of 3 mm of the distal part of the nerve was cut away. The length of the nerve used was 4 cm. The left hemidiaphragm was then taken out, the dorsal slip being discarded. The muscle was carefully cleaned, washed in saline, pressed between gauze pads and weighed. The weight of the acetone dried powder
527
INCREASE IN CHAC ACTIVITY
was used as dry weight. In this type of experiment 20 rats were used; 10 were control litter mates to the denervated ones. Estimation of choline acetyltransferase actiuity. The method devised by Hebb (see Nordenfelt 1963, 1965 a) was used. Acetone dried powder was prepared from individual salivary glands, except for the sublingual glands where 4 or 5 glands were pooled. Acetone dried powder was also made from individual hemidiaphragms; 2 phrenic nerves were pooled. The powder of the glands and the hemidiaphragms was made up in cysteine-saline in a concentration of 50 mg/ml, the powder of the phrenic nerves in a concentration of 12 cm nerve/ml. Of the tissue extracts from the glands and the nerves 0.2 ml, from the hemidiaphragms 0.4 ml, were incubated at 38°C for 60 min. The incubate was assayed for acetylcholine on the eserinized frog rectus. The choline acetyltransferase activity is expressed in p g acetylcholine chloride formed pr h per gland or pooled glands (total activity) and in p g acetylcholine chloride formed per h per g acetone powder or per cm of nerve (concentration). Student’s t-test was used; paired comparison were made between the operated rat and its control litter mate. P values of less than 0.05 were considered significant.
Results Body-weights. At the start of the experiments no differences existed in body-weights between the rats to be used as controls and those to be operated on. When the final body-weights of the rats are expressed in per cent of their initial weights the mean percentage figure obtained in the duct-ligated animals 3 days after the beginning of the experiment was 3 YUlower (p
2-
5
EQ-
8
40-
9
co.01
-
100-
b
80-
$
120-
p i
20-
n.5
n-9
n.5
3DAYS
?DAYS 28DAYS I PAnOTlD
n-9 28DAYS
SUBMAXILLARY
I
8060-
40-
20-
% ! -
n -10 28 DAYS DIAPHRAGM
n.4 28 DAYS
PHRENE NERVE
2
Fig. I . Dry weight and choline acetyltransferase activity (calculated per gland) of the intact salivary glands at different time intervals after the ligation procedure. The weight and the enzyme activity are expressed as a percentage of those of the gland of the control litter mate (mean +S.E.). Number of comparisons are shown and when the differences are significant the p-values are given. Fig. 2. Dry weight and choline acetyltransferase activity (calculated per hemidiaphragm) of the intact half of the diaphragm muscle and choline acetyltransferase activity of the phrenic nerve (calculated per pool of 2 nerves) after section of the nerve on the contralateral side. Results are expressed as in Fig. 1.
rats was 45 -t 1 breaths per 30 s. The enzyme activity of the left phrenic nerve in the rats, in which the nerve on the right side had been sectioned 28 days earlier, did not differ from that of the nerve in the control rats, as shown in Fig. 2. However, the total enzyme activity was 13% higher in the left hemidiaphragm than it was in the control muscle; no change in the dry weight was observed. The respiratory frequency of the operated rats was found to be higher (p -.0.001) than that of the control rats when expressed in per cent of the controls it was 131 + 3 (n - 10).
Discussion The present study shows that after 4 weeks of increased impulse traffic in efferent pathways the choline acetyltransferase activity is increased in salivary glands and in hemidiaphragm. In the earlier study, in which incrsased reflex stimulation of salivary glands also was induced, increased enzyme activity was at hand when the glands were examined 3 weeks after the beginning of the experiments (Ekstrom 1974). It appears from the present investigation that it takes some time for the phenomenon to develop, since no increase in the enzyme activity was observed in the parotid gland within the first week. The secretory activity of the gland was probably increased already at the end of this week, as shown by the glandular hypertrophy. It is of interest to note that the increase in choline acetyltransferase activity produced in some salivary glands by sympathetic denervation also seems to be a late phenomenon, not occurring until after about 2 weeks (Nordenfelt 1965 b, Ekstrorn 1972). A possible ex-
INCREASE IN CHAC ACTIVITY
529
planation to the fact that the increase in enzyme activity failed to appear in the skeletal muscle studied by Diamond et al. (1974) might be that the time period chosen was too short to allow the increase to develop under their experimental conditions. The increase in choline acetyltransferase activity was bigger in the parotid gland than in the hemidiaphragm muscle. Although it is possible to obtain long pieces of the auriculotemporal nerve for choline acetyltransferase analysis, the disadvantage with the nerve is its early branching. Therefore, in the present work the phrenic nerve was used. No change in the enzyme activity was observed; the activity in the nerve was of the same order as that of earlier investigations (Hebb et al. 1964, Ekstrom and Emmelin 1971). It thus appears that the increase in activity of the acetylcholine synthesizing enzyme is particularly manifested in the nerve endings. Mucosal dryness and dry food were in the previous work found to affect especially the parotid gland, as judged from the increase in choline acetyltransferase activity and the increase in weight (Ekstrom 1974). It is also this gland which shows the biggest glandular enlargement in the present study. No hypertrophy was found in the hemidiaphragm; this is in accordance with the observation of Sola and Martin (1953). This work was supported by a grant from the Faculty of Medicine in Lund.
References DIAMOND, I., G. M. FRANKLIN and D. MILFAY,The relationship of choline acetyltransferase activity at the neuromuscular junction to changes in muscle mass and function. J. Physiol. (Lond.) 1974. 236. 247-257. J . , Choline acetyltransferase in salivary glands after surgical and chemical sympathectomy. Acra EKSTROM, physiol. scand. 1972. 86. 539-545. J . , Choline acetyltransferase and secretory responses of the rat’s salivary glands after liquid diet. EKSTROM, Quart. J. exp. Physiol. 1973. 58. 17 1-1 79. J., Choline acetyltransferase activity in rat salivary glands after cellulose rich diet or treatment EKSTROM, with an atropine-like drug. Quart. J. exp. Physiol. 1974. 59. 191-199. Movement of choline acetyltransferase in axons disconnected from their EKSTROM,J. and N. EMMELIN, cell bodies. J. Physiol. (Lond.) 1971. 216. 247-256. J . and J. HOLMBERG, Effect of decentralization on the choline acetyltransferase of the canine EKSTR~M, parotid gland. J. Physiol. (Lond.) 1972 a. 222. 93-94 P. EKSTR~M J. , and J. HOLMBERG, Choline acetyltransferase in the normal and parasympathetically denervated parotid gland of the dog. Acra physiol. scand. 1972 b. 86. 353-358. ELMER,M. and P. OHLIN,Compensatory hypertrophy of the rat’s submaxillary gland. Acta physiol. scand. 1969. 76. 396-398. EMMELIN, N. and L. MALM,Development of supersensitivity as dependent on the length of degenerating nerve fibres. Quarr. J. exp. Physiol. 1965. 50. 142-145. and C. PEREC,Rate of fall in choline acetyltransferase activity in denervated EMMELIN, N., 1. NORDENFELT diaphragms as dependent o n the length of the degenerating nerve. Experienriu (Basel) 1966. 22. 725. K V. HANZLLKOVA, Changes in the choline acetyltransferase and cholinesterase GUTMANN, E., S. T U ~ Eand activities in the levator ani muscle of rats following castration. Physiol. bohemoslou. 1969. 18. 195-203. and A. SILVER,Acetylcholine and choline acetyltransferase in the diaphragm of HEBB,C . O., K. KRNJEVIC the rat. J. Physiol. (Lond.) 1964. 171. 504-513. ISRABL,M., Localisation de I’ac6tylcholine des synapses myoneurales et nerf-8lectroplaque. Arrh. Anar. micr. Morph. exp. 1970. 59. 67-98. I., Choline acetylase in normal and denervated salivary glands. Quarr. J. exp. Physiol. 1963. NORDENFELT, 48. 61-19. 34 - 155874
530
JORGEN E K S T R ~ M
NORDENFELT, I., Effect of preganglionic parasympathetic denervation on choline acetylase in postganglionic neurones. Acta Uniu. Lund. 11. 1964. 16. 1-7. NORDENFELT, I., Acetylcholine synthesis in saliuary glands. Lund. Hhkan Ohlsson, 1965 a. (Thesis). NORDENFELT, I., Choline acetylase in salivary glands of the cat after sympathetic denervation. Quart. J. exp. Physiol. 1965 b. 50. 57-61. SOLA,0. M. and A. W. MARTIN. Denervation hypertrophy and atrophy of the hemidiaphragm of the rat. Amer. J. Physiol. 1953. 172. 324-332. SNYDER, D. H., D. H. RIFENEERICK and S. R. MAX,Effects of neuromuscular activity on choline acetyltransferase and acetylcholinesterase. Exp. Neurol. 1973. 40. 36-42. WELL^, H. and A. A. V. PERONACE, Synergistic autonomic nervous regulation of accelerated salivary gland growth in rat. Amer. J. Physiol. 1964. 207. 313-318.
Compensatory Increase in Choline Acetyltransferase Activity in Salivary Glands and Diaphragm Muscle of the Rat BY JORGEN
EKSTROM
Received 29 October 1974
Abstract EKSTROM, J. Compensatory increase in choline acetyltransferase activity in salivary glands and diaphragm muscle of the rat. Acta physiol. scand. 1975. 93. 525-530. When the function of salivary glands was abolished by applying ligatures to their ducts and the function of one half of the diaphragm muscle was abolished by sectioning of its phrenic nerve, the choline acetyltransferase activity was found to be increased in not duct-ligated glands and in the intact hemidiaphragm 4 weeks later. The increase was not seen within the first week. The increase in activity appears to be particularly manifested in the nerve endings, since it was seen in the hemidiaphragm but not in the phrenic nerve. Increased stream of impulses in the efferent nerves is thought to be the cause of this increase in choline acetyltransferase activity. Key words: Choline acetyltransfeidse activity, salivary glands, diaphragm muscle
When the preganglionic parasympathetic salivary nerve is cut the ability to form acetylcholine in the postganglionic neurones of the gland decreases, as judged from the reduction of choline acetyltransferase activity (Nordenfelt 1964, Ekstrom and Holmberg 1972 a). A fall in the activity of this enzyme of about the same magnitude can be induced in the parotid gland of the rat by keeping the animal on a liquid diet (Ekstrom 1973). The opposite, i.e. a rise in the enzyme activity occurs in the gland when the rat is fed on a dry, cellulose rich diet or when the animal is atropinized causing dryness of the oral mucosa (Ekstrom 1974). The results of these studies suggest that the choline acetyltransferase activity is depending on the impulse-propagating activity of the neurones, as originally pointed out by Nordenfelt (1964); the enzyme activity declines when the flow of secretory impulses in the neurones is lost or reduced and it increases when the impulse traffic is augmented. Observations on skeletal muscles show that disuse is followed by a decrease in choline acetyltransferase activity (Gutmann, TuEek and HanzlikovB 1969, Snyder, Rifenberick and Max 1973, Diamond, Milfay and Franklin 1974). However, opinions differ as to the effect of overuse. Thus an increased choline acetyltransferase activity was found by Snyder et al. (1973) in the plantaris muscle of the rat, when the tendon of the synergistic muscles, gastroc525
526
JORGEN EKSTROM
nemius and soleus, had been severed for 8 days, while Diamond et al. (1974) were unable to demonstrate any increase in the rat gastrocnemius muscle 7 days after the tendons of the soleus and plantaris muscles had been cut. When saliva is prevented from reaching the oral cavity of the rat by ligation of the ducts of some of the salivary glands the reflexly elicited stimulation of the other not duct-ligated glands appears to be augmented, as judged from glandular enlargement (Wells and Peronace 1964, Elmer and Ohlin 1969) and increased secretion (Elm& and Ohlin 1969). In the present study the choline acetyltransferase activity was estimated in such non-ligated glands. As in salivary glands (see Nordenfelt 1965 a, Ekstrom and Holmberg 1972 b) the acetylcholine forming enzyme is in the diaphragm muscle confined to the cholinergic neurones (Hebb, KrnjeviC and Silver 1964, Emmelin, Nordenfelt and Perec 1966, Israel 1970). In the present investigation the enzyme activity was determined in one half of the muscle and in its phrenic nerve when the nerve on the contralateral side of the rat had been sectioned in advance. This procedure seems to lead to an increase in the stimulation of the innervated hemidiaphragm since, according to the result of preliminary experiments, the respiratory frequency of the rat rose.
Methods 74 male rats of a Sprague-Dawley strain bred at this Institute were used. The rats were at the start of the experiments 3-5 months old and weighed 260-380 g. They were given a standard pelleted diet and water ad libitum. The experiments were performed as follows: Sa/iiwr.v glands. The rats were anaesthetized with ether. In the first series the parotid duct on the left side and the ducts of the submaxillary and the sublingual glands on both sides were tied with a fine silk thread, the ducts of the latter two glands close to hilus causing at the same time an interruption of the parasympathetic nerve supply. In the second series both parotid ducts and those of the submaxillary and the sublingual glands on the left side were ligated. At the end of the experimental period the rats were killed with ether and weighed. The position of the ligatures was controlled and macroscopical changes of the ductligated glands were looked for, such as dark red colour and atrophy. In the first series of experiments the nonligated right parotid glands from 5 rats were examined 3 days after the ligation procedure, glands from another 5 rats 4 days later, and from 9 rats 28 days after the beginning of the investigation. In the second series the submaxillary and the sublingual glands on the right side of 9 rats were studied after 28 days, too. When the glands had been removed, they were cleaned, washed in saline, pressed gently between gauze pads and weighed. Since dry weight is considered more reliable, the weight of the acetone dried powder was used. As control glands served the corresponding ones of litter mates to duct-ligated rats; in two cases the control rat was common to both the series of experiments. Thus 54 rats were used. Phrenic neriv and diaphragm. The rats were anaesthetized with pentobarbitone, 60 mg/kp i.p. At the same time atropine sulphate, 1 mg/kg i.p., was given in order t o suppress bronchial secretion. Artificial respiration was given by a pump through a fine glass tube which was inserted into the trachea through a small cut. Between 2 ribs the right phrenic nerve was sectioned in the upper part of the thorax and a nerve segment of about 5 mm was removed. The intrathoracic approach was chosen, since some of the cervical branches forming the phrenic nerve otherwise might escape the section when done in the neck region (Sola and Martin 1953, Emmelin and Malm 1965). The wounds were sutured and the artificial respiration was stopped. After 28 days the respiratory frequency of the operated rat was estimated, when the animal seemed to be at rest; the frequency was also determined in the litter mate used as control. The rats were then killed with ether and weighed. The right sectioned phrenic nerve was inspected. In some instances signs of beginning nerve regeneration could be observed in the form of anatomical connection with the proximal part of the phrenic nerve or intercostal nerves. The left phrenic nerve was cut close to the diaphragm and taken out. A piece of 3 mm of the distal part of the nerve was cut away. The length of the nerve used was 4 cm. The left hemidiaphragm was then taken out, the dorsal slip being discarded. The muscle was carefully cleaned, washed in saline, pressed between gauze pads and weighed. The weight of the acetone dried powder
527
INCREASE IN CHAC ACTIVITY
was used as dry weight. In this type of experiment 20 rats were used; 10 were control litter mates to the denervated ones. Estimation of choline acetyltransferase actiuity. The method devised by Hebb (see Nordenfelt 1963, 1965 a) was used. Acetone dried powder was prepared from individual salivary glands, except for the sublingual glands where 4 or 5 glands were pooled. Acetone dried powder was also made from individual hemidiaphragms; 2 phrenic nerves were pooled. The powder of the glands and the hemidiaphragms was made up in cysteine-saline in a concentration of 50 mg/ml, the powder of the phrenic nerves in a concentration of 12 cm nerve/ml. Of the tissue extracts from the glands and the nerves 0.2 ml, from the hemidiaphragms 0.4 ml, were incubated at 38°C for 60 min. The incubate was assayed for acetylcholine on the eserinized frog rectus. The choline acetyltransferase activity is expressed in p g acetylcholine chloride formed pr h per gland or pooled glands (total activity) and in p g acetylcholine chloride formed per h per g acetone powder or per cm of nerve (concentration). Student’s t-test was used; paired comparison were made between the operated rat and its control litter mate. P values of less than 0.05 were considered significant.
Results Body-weights. At the start of the experiments no differences existed in body-weights between the rats to be used as controls and those to be operated on. When the final body-weights of the rats are expressed in per cent of their initial weights the mean percentage figure obtained in the duct-ligated animals 3 days after the beginning of the experiment was 3 YUlower (p
2-
5
EQ-
8
40-
9
co.01
-
100-
b
80-
$
120-
p i
20-
n.5
n-9
n.5
3DAYS
?DAYS 28DAYS I PAnOTlD
n-9 28DAYS
SUBMAXILLARY
I
8060-
40-
20-
% ! -
n -10 28 DAYS DIAPHRAGM
n.4 28 DAYS
PHRENE NERVE
2
Fig. I . Dry weight and choline acetyltransferase activity (calculated per gland) of the intact salivary glands at different time intervals after the ligation procedure. The weight and the enzyme activity are expressed as a percentage of those of the gland of the control litter mate (mean +S.E.). Number of comparisons are shown and when the differences are significant the p-values are given. Fig. 2. Dry weight and choline acetyltransferase activity (calculated per hemidiaphragm) of the intact half of the diaphragm muscle and choline acetyltransferase activity of the phrenic nerve (calculated per pool of 2 nerves) after section of the nerve on the contralateral side. Results are expressed as in Fig. 1.
rats was 45 -t 1 breaths per 30 s. The enzyme activity of the left phrenic nerve in the rats, in which the nerve on the right side had been sectioned 28 days earlier, did not differ from that of the nerve in the control rats, as shown in Fig. 2. However, the total enzyme activity was 13% higher in the left hemidiaphragm than it was in the control muscle; no change in the dry weight was observed. The respiratory frequency of the operated rats was found to be higher (p -.0.001) than that of the control rats when expressed in per cent of the controls it was 131 + 3 (n - 10).
Discussion The present study shows that after 4 weeks of increased impulse traffic in efferent pathways the choline acetyltransferase activity is increased in salivary glands and in hemidiaphragm. In the earlier study, in which incrsased reflex stimulation of salivary glands also was induced, increased enzyme activity was at hand when the glands were examined 3 weeks after the beginning of the experiments (Ekstrom 1974). It appears from the present investigation that it takes some time for the phenomenon to develop, since no increase in the enzyme activity was observed in the parotid gland within the first week. The secretory activity of the gland was probably increased already at the end of this week, as shown by the glandular hypertrophy. It is of interest to note that the increase in choline acetyltransferase activity produced in some salivary glands by sympathetic denervation also seems to be a late phenomenon, not occurring until after about 2 weeks (Nordenfelt 1965 b, Ekstrorn 1972). A possible ex-
INCREASE IN CHAC ACTIVITY
529
planation to the fact that the increase in enzyme activity failed to appear in the skeletal muscle studied by Diamond et al. (1974) might be that the time period chosen was too short to allow the increase to develop under their experimental conditions. The increase in choline acetyltransferase activity was bigger in the parotid gland than in the hemidiaphragm muscle. Although it is possible to obtain long pieces of the auriculotemporal nerve for choline acetyltransferase analysis, the disadvantage with the nerve is its early branching. Therefore, in the present work the phrenic nerve was used. No change in the enzyme activity was observed; the activity in the nerve was of the same order as that of earlier investigations (Hebb et al. 1964, Ekstrom and Emmelin 1971). It thus appears that the increase in activity of the acetylcholine synthesizing enzyme is particularly manifested in the nerve endings. Mucosal dryness and dry food were in the previous work found to affect especially the parotid gland, as judged from the increase in choline acetyltransferase activity and the increase in weight (Ekstrom 1974). It is also this gland which shows the biggest glandular enlargement in the present study. No hypertrophy was found in the hemidiaphragm; this is in accordance with the observation of Sola and Martin (1953). This work was supported by a grant from the Faculty of Medicine in Lund.
References DIAMOND, I., G. M. FRANKLIN and D. MILFAY,The relationship of choline acetyltransferase activity at the neuromuscular junction to changes in muscle mass and function. J. Physiol. (Lond.) 1974. 236. 247-257. J . , Choline acetyltransferase in salivary glands after surgical and chemical sympathectomy. Acra EKSTROM, physiol. scand. 1972. 86. 539-545. J . , Choline acetyltransferase and secretory responses of the rat’s salivary glands after liquid diet. EKSTROM, Quart. J. exp. Physiol. 1973. 58. 17 1-1 79. J., Choline acetyltransferase activity in rat salivary glands after cellulose rich diet or treatment EKSTROM, with an atropine-like drug. Quart. J. exp. Physiol. 1974. 59. 191-199. Movement of choline acetyltransferase in axons disconnected from their EKSTROM,J. and N. EMMELIN, cell bodies. J. Physiol. (Lond.) 1971. 216. 247-256. J . and J. HOLMBERG, Effect of decentralization on the choline acetyltransferase of the canine EKSTR~M, parotid gland. J. Physiol. (Lond.) 1972 a. 222. 93-94 P. EKSTR~M J. , and J. HOLMBERG, Choline acetyltransferase in the normal and parasympathetically denervated parotid gland of the dog. Acra physiol. scand. 1972 b. 86. 353-358. ELMER,M. and P. OHLIN,Compensatory hypertrophy of the rat’s submaxillary gland. Acta physiol. scand. 1969. 76. 396-398. EMMELIN, N. and L. MALM,Development of supersensitivity as dependent on the length of degenerating nerve fibres. Quarr. J. exp. Physiol. 1965. 50. 142-145. and C. PEREC,Rate of fall in choline acetyltransferase activity in denervated EMMELIN, N., 1. NORDENFELT diaphragms as dependent o n the length of the degenerating nerve. Experienriu (Basel) 1966. 22. 725. K V. HANZLLKOVA, Changes in the choline acetyltransferase and cholinesterase GUTMANN, E., S. T U ~ Eand activities in the levator ani muscle of rats following castration. Physiol. bohemoslou. 1969. 18. 195-203. and A. SILVER,Acetylcholine and choline acetyltransferase in the diaphragm of HEBB,C . O., K. KRNJEVIC the rat. J. Physiol. (Lond.) 1964. 171. 504-513. ISRABL,M., Localisation de I’ac6tylcholine des synapses myoneurales et nerf-8lectroplaque. Arrh. Anar. micr. Morph. exp. 1970. 59. 67-98. I., Choline acetylase in normal and denervated salivary glands. Quarr. J. exp. Physiol. 1963. NORDENFELT, 48. 61-19. 34 - 155874
530
JORGEN E K S T R ~ M
NORDENFELT, I., Effect of preganglionic parasympathetic denervation on choline acetylase in postganglionic neurones. Acta Uniu. Lund. 11. 1964. 16. 1-7. NORDENFELT, I., Acetylcholine synthesis in saliuary glands. Lund. Hhkan Ohlsson, 1965 a. (Thesis). NORDENFELT, I., Choline acetylase in salivary glands of the cat after sympathetic denervation. Quart. J. exp. Physiol. 1965 b. 50. 57-61. SOLA,0. M. and A. W. MARTIN. Denervation hypertrophy and atrophy of the hemidiaphragm of the rat. Amer. J. Physiol. 1953. 172. 324-332. SNYDER, D. H., D. H. RIFENEERICK and S. R. MAX,Effects of neuromuscular activity on choline acetyltransferase and acetylcholinesterase. Exp. Neurol. 1973. 40. 36-42. WELL^, H. and A. A. V. PERONACE, Synergistic autonomic nervous regulation of accelerated salivary gland growth in rat. Amer. J. Physiol. 1964. 207. 313-318.
