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Effect of denervation on muscle cyclic AMP in the newt
ESPERIMEKTAL
XECROLOGY
49, 716-724 (1975)
Effect of Denervation
on Muscle Cyclic AMP in the Newt THOMAS
Section
of
Cytology, Yale New Haven, Received
L. LENTZ~ University Corm&cut June
School 06510
of
Medicine,
21, 1975
The effects of denervation ir, z&o and of nerve extracts in vitro on muscle cyclic AMP levels were measured to determine whether nerves influence muscle cyclic AMP. Denervation in viz10 resulted in an initial small decrease followed by a large increase in muscle cyclic AMP. Cyclic AMP levels declined as the muscle became reinnervated. After brief times in culture (l-3 days), cyclic AMP levels were lower in untreated muscles than in those cultured in the presmence of nerve extracts. At longer times, cyclic AMP was the same or higher in untreated muscles.
INTRODUCTION In addition to the short-term changes associated with transmission of the nerve impulse, the motor neuron exerts several long-term regulatory influences, defined as “trophic,” on the molecular specializations of the skeletal muscle fiber. One such influence is the neural regulation of muscle cholinesterase (ChE) activity, which is induced in developing or regenerating muscle by the nerve fiber and is reduced following denervation (3, 6-8, 11). The sensitivity of muscle ChE to neural influences has been utilized in an ill vitro system to assay potential trophic factors (9, 13, 17). The assay tests the ability of substances or experimental treatments to prevent the decreasein ChE activity of cultured skeletal muscle occurring as a result of denervation. Utilizing this system, it has been shown that nerve explants and extracts are effective in maintaining ChE activity of cultured muscle. Furthermore, adenosine 3’S’-cyclic monophosphate (cyclic AMP) in combination with theophylline, an inhibitor of cyclic nucleotide phospho1 Supported by a grant (GB-20902) from the National Science Foundation. I would like to thank Dr. James Nathanson, Ms. Linda Brown, and Ms. Janice Chester for assistance in the experimental procedures and Dr. Paul Greengard for reading the manuscript. 716 Copyright 0 1975 by Academic Press, Inc. All rights of repwduction in any form reserved.
CYCLIC
AMP
717
diesterase, and dibutyryl cyclic AMP have been found to have an effect similar to that of nerve explants in maintaining the ChE activity of denervated muscle in organ culture (IO, 1::7 IS). In addition, cyclic AMP has been implicated in another trophically regulated system, the regenerating limb of the newt, where it stimulates mitotic activity and regenerative growth (4). The action of cyclic A?IIP in mediating the effects of polypeptide hormones is well known. In addition, evidence has been presented recently that the actions of some neurotransmitters are mediated by cyclic AMP (5, 20). It has been hypothesized by McVahon (15) that cyclic nucleotides, under the regulatory influence of inducers which may include neurotransmitters, are involved in the regulation of developmental processes throughout the eukaryotes. In view of evidence that in certain situations neurons can exert postsynaptic effects on cyclic AMP levels, and the observation that cyclic AMP mimics the effect of nerve on muscle ChE, it seems possible that the trophic effects of nerve on muscle might be mediated by cyclic AMP formed in response to the trophic influence (10). If such trophic effects are mediated by cyclic AMP, the motor neuron should exert regulatory influences on muscle adenylate cyclase or possibly phosphodiesterase activities resulting in alterations of muscle cyclic AMP levels. The present study was carried out to measure the effects of denervation in zlizfo and of nerve extracts irr zifvo on cyclic AMP levels of muscle to determine whether innervation influences muscle cyclic AMI’. MATERIALS
AKD
METHODS
These studies were performed on the newt Trifunrs zh2escens. Cyclic AMP was measured in muscles of normal animals, after denervation, after tenotomy, after elimination of the blood supply to the limb, and in muscles placed in organ culture and treated with nerve extracts. In all cases,cyclic AMP was measured in paired forelimb muscles, one of which was treated experimentally, while the other served as an untreated control. Five to 1S pairs of muscle were used in each experiment. Operative procedures were performed on newts anesthetized in O.Zc/u chloretone. Denervation was achieved in one forelimb by exposing the hrachial plexus and remoGng a 1 mm segment of the brachial nerve cords. This procedure eliminates the motor and sensory supply (nerves 3, 4, and 5) to the limb. A sham operation was performed on the opposite limb, and the nerves left intact. The triceps muscles from the denervated limb and the innervated opposite limb were removed at various intervals for assay of cyclic ;\nrlj content. At various times. the brnchial plexus and triceps muscles of tlcnervatetl animals \vere exposed and examined
718
THOMAS
L.
LENT2
under a dissecting microscope to follow the time course of reinnervation of the limb. Tenotomy was accomplished by cutting the tendinous insertions and origins of the triceps muscle. The blood supply to the limb was eliminated by pinching and severing the brachial vessels. Muscles were cultured as described previously (9). One muscle of each pair was cultured in media containing newt brain extract prepared as previously described (13). Concentrations of extract known to maintain cholinesterase activity were used. The effect of liver extract on cyclic AMP was also tested. For cyclic AMP assay, muscles were removed and homogenized with a glass homogenizer in 1 ml cold 985% ethanol-0.2N HCl. If the muscles were small, two or three were pooled. The homogenate was centrifuged at 9,000 rpm in an International bench model centrifuge. Two 0.3 ml aliquots of each sample were removed and dried for cyclic AMP assay. The precipitate was assayed for protein with the method of Lowry et al. (14). The amount of cyclic AMP in each aliquot was measured by the method of Brown, Ekins, and Albano (1). Cyclic AMP content was calculated from a linear standard curve constructed with from 0.1 to 1.4 pmole of cyclic AMP. The amount of cyclic AMP per muscle was determined and divided by the protein content of each muscle to give pmole cyclic AMP per milligram of protein, RESULTS Individual newts differ in muscle cyclic AMP content; however, cyclic AMP content of triceps muscles from the same animal is the same (Table 1) . Thus, by utilizing pairs of muscles from the same animal, the samples are paired or nonindependent and the differences between members of pairs can be treated as single observations. In Tables 1 and 2, mean values for cyclic AMP levels are shown for control muscles, experimental muscles, and the differences between the two. The standard error of the mean (SEM) is given for the differences which are tested by the t distribution at the 0.05 or 0.01 significance levels. Cyclic AMP levels of denervated muscle undergo a relatively small, but significant decline in the first 2 days after nerve transection (Table 1, Fig. 1) . Thereafter, cyclic AMP rises to levels considerably higher than in the normally innervated muscles of the same animals reaching a maximum at 3 to 4 weeks. At this time, the denervated muscles are grossly smaller and atrophied. Cyclic AMP levels then begin to decline as the muscles are reinnervated, enlarge, and resume function. In tenotomized muscles, cyclic AMP undergoes a rapid and large increase. A transient decrease such as occurs following denervation did not occur after tenotomy. Cutting
Blood
muscles
vessels
Tenotomy
Denervation
Normal
Procedure
cut
Id
Id 16d
lh 4h Id 2d 3d 4d 8d 16d 24d 49d
Time
6
6 7
14 12 16 7 12 11 14 9 6 7
13
Number pairs
of
EFFECT
TABLE
1.50
Intact
2.07 2.65
Intact
1.99 2.83 3.17 2.78 2.03 3.27 1.92 2.33 1.68 4.00
Sham
3.64
1
3.62
Left
OS MUSCLE
1.53
cut
2.34 6.70
cut
1.90 2.61 2.32 2.07 1.83 3.52 1.93 2.84 3.81 6.09
Denervated
(pmol/mg protein)
Right
CAMP
OF DENERVATION
0.02
0.03
0.27 4.05
f
f f
f f f f f f f f f zk
f
0.07
0.12 0.33
0.21 0.25 0.33 0.28 0.16 0.37 0.21 0.24 0.19 0.70
0.35
of differences +SEM
-0.09 -0.23 -0.85 -0.71 -0.21 -0.26 0.01 0.51 2.13 2.09
Mean
CAMP
>o.os
>o.os
>o.os >o.os <0.0.5o.os >o.os >0.0.5 >o.os
>o.os
P
+2
+13 +153
+127 +59
+22
-8 -27 -26 -10 -7 +1
-5
-1
Difference G)
5
w
720 the blood vessels to the 1iIlllJ (lit1 not affect cyclic i\lll’ Icvels at 1 day. Thus, the changes in cJ.clic :jJl I’. at least at this time, are not the result of possible interference of the blootl supply by the (Jpxdve procedures. At longer times after blood vessel section, the muscles became necrotic so that it was not possible to measure cyclic L1hlP. The differences between untreated nluscles and muscles exposed to nerve extract ilz. z&o followed a l)attern similar to the differences between denervated and innervated mukcles ill zko during the first 3 days (Table 3). After 1 to 3 days in culture, cyclic AMP levels in untreated (comparable to denervated) muscles were lower than in those cultured in the presence of nerve estracts, although it appears that cyclic AMP decreases in both treated and untreated muscle in culture. After 8 days in culture, cyclic AMP was the same or greater in the untreated mu4e than in the treated muscle. Liver extract had no effect on muscle cyclic AMP. DISCUSSION It was found in the newt that loss of innervation results in an initial small decrease followed by a large and prolonged rise in muscle cyclic AMP levels. These findings indicate the motor nerve fiber may exert some control on the cyclic AMP levels of skeletal muscle. The significance of the observed changes in relation to trophic mechanisms is difficult to determine, however. The initial decrease in cyclic AMP in denervated muscle is consistent with the hypothesis that trophic effects might be mediated by cyclic AMP formed in response to the neuronal influence. In addition, cyclic AMP levels ia z&o could be maintained for 1 to 3 days by brain extracts. Brain extracts have also been found to stimulate accumulation of cyclic AMP in slices of cerebral cortex after very brief exposure (19). On the other hand, the large subsequent increase in cyclic AMP levels in denervated muscle as well as the lower levels in muscle treated with nerve extracts in longer term cultures, seem to indicate innervation might normally suppress cyclic AMP levels. Carlsen (2)) in studying cyclic AMP changes in denervated rat gastrocnemius, found that cyclic AMP levels increased following denervation and were reduced with reinnervation. These changes were similar to those described in the present study, although an initial increase in cyclic AMP was not observed and the changes occurred much more rapidly in the rat than in the newt. Carlsen (2) found in addition that the onset of the postdenervation increase in cyclic AMP depended on the length of the nerve stump. He suggested that muscle activity might suppress cyclic AMP, but that, in addition, a chemical factor supplied by the nerve regulates the cyclic AMP system.
Brain Brain Brain Brain Brain Liver
Extract
(0.07 (0.31 (0.07 (0.31 (0.31 (0.07
mg/ml) mg/ml) mg/mI) mg/ml) mg,/ml) mg/ml)
(mg protein,‘ml medium)
__-
1 1 3 3 8 3
Time (days)
6 12 18 12 12 5
Number pairs
of
NERVETISSVE
CAMP
1.88 2.14 0.85 0.84 0.63 1.86
Plain medium
-.
TABLE
2
2.25 2.36 1.68 1.10 0.54 1.93
Medium fextract
(pmol,‘mg protein)
EXTRACT-EFFECTOS
* Lt f i * zk
0.15 0.05 0.19 0.14 0.24 0.56
of differences GEM
CAMP
0.37 0.22 0.83 0.27 -0.09 0.07
Mean
MUSCLE
_---.-
P-o.050.05 >o.os >o.os
P
+20 +10 +97 +32 -15 +4
Difference (yy) si p. c c, > R 9
722
TIIOMAS
L.
LENT2
-so
FIG. 1. Effect of denervation and tenotomy on cyclic AMP levels in the triceps muscle of the newt. Denervation of muscle results in an initial decrease followed by a large increase in muscle cyclic AMP. Tenotomy produces a large increase in cyclic AMP. See Table 1 for data and statistics.
Caution is required, however, in drawing conclusions regarding neural influences on cyclic AMP solely on the basis of cyclic AMP measurements, since there can be increased turnover of cyclic AMP and a physiological effect without accumulation or even with a decline in total cyclic AMP during a hormonally regulated process. In addition, it is probable that a number of factors influence cyclic AMP levels of muscle, and these effects must be distinguished from those of nerve. The effects of tenotomy, for example, might indicate that muscle activity has an influence on cyclic AMP, acting to suppress it. The large increase in cyclic AMP in denervated muscle could, therefore, be the result of muscle inactivity following denervation. It should be noted, however, that in the cat, tenotomy of a single muscle results in little or no change in electromyographic activity (16). It appears at this point that denervation results in alterations in muscle cyclic AMP levels and that cyclic AMP plays a role in the maintenance of at least one of the differentiated characteristics (ChE) of the muscle fiber which is also dependent on innervation. The relation between innervation and cyclic AMP and the mechanism of action of cyclic AMP
remain unclear. Further information on the action of nerves in regulation of muscle cyclic AMP could be obtained by determining the effect of neural inAuences on muscle adenyl cyclase, phosphodiesterase, and protein kinases and their substrates.
REFERENCES B. L., R. P. EMNS, and J. U. $1. ALBANO. 1972. Saturation assay for cyclic-AMP using endogenous binding protein. .-idv. Cyclic X~ccleotidz Krs. 2: 25-40. CARLSEN, R. C. 1975. The possible role of cyclic AMP in the neurotrophic control of skeletal muscle. J. Physiol. (Lor~don) 247 : 343-361. ER;~NKO, O., and H. TEKXVXINEN. 1967. Cholinesterases and eserine-resistant carboxylic esterases in degenerating and regenerating motor end plates of the rat. J. ~Vfttrockcw~. 14: 947-957. FORET, J. E. 1973. Stimulation and retardation of limb regeneration by adenosine 3’,5’-monophosphate and related compounds. OHC(J/O~J~ 27 : 153-159. GREENGAHD, P., D. A. MCAFEE, and J. W. KEBABIAX. 1972. On the mechanism of action of cyclic AMP and its role in synaptic transmission. .dd;a. Cyc-/il. Nucleotide Rrs. 1 : 373-390. GUTH, L., and W. C. BROWS. 1965. The sequence of changes in cholinesterase activity during reinnervation of muscle. Exp. Llrczrrol. 12: 329-336. GUTII, L.. R. W. ALBEES, and W. C. BROWVN. 1964. Quantitative changes in cholinesterase activity of denervated muscle fihers and sole plates. Krb. .Vcrtrol. 10: 236-250. HALL, Z. W. 1973. Multiple forms of acetylcholinesterase and their distribution in endplate and non-endplate regions of rat diaphragm muscle. J. Srrrro6iol. 4: 343361. LENTZ, T. L. 1971. Nerve trophic function. I>r z~itro assay of effects of nerve tissue on muscle cholinesterase activity. S&m-c 171 : 187-189. LENTZ, T. L. 1972a. A role of cyclic .4MP in a neurotrophic process. ,Vtrturc (Nczo Viol.) 238 : 154-15.5. LENTZ, T. L. 1972b. Development of the neuromuscular junction. III. Degeneration of motor end plates after denervation and maintenance irt critro hy nerve explants. J. Cell Lliol. 55: 93-103. LENTZ, T. L. 1974a. Neurotrophic regulation at the neuromuscular junction, An11. N. Y. Acad. Sci., 228: 323-337. LENTZ, T. L. 1974b. Effect of brain extracts on cholinesterase activity of cultured skeletal muscle. E.rp. Xmrol. 45: 520-51’6. LOWRY, 0. H., N. J. ROSEEKOCGH, A. L. FARR, and R. J. RANDALL. 1951. Protein measurement with the Folin phenol reagent. J. Riol. Cltcur. 193: 265-275. MCMAHON, D. 1974. Chemical messengers in development: A hypothesis, .‘?<.iracc 185 : 1012-1021. NELSON, P. G. 1969. Functional consequences of tenotomy in hitILl limh muscles of the cat. 1. Physiol. (London) 201: 321-333. OH, T. H., D. D. JOHNSON, and S. U. KIM. 1972. Neurotrophic effect on isolated chick embryo muscle in culture. Scirrzrc 178: 1298-1300.
1. BROWN,
I’.
3.
4. 5.
6. 7.
8.
9. 10. 11.
12.
13. 14. 15. 16. 17.
724 1X. RATHBOPITE, M. P., factor from chick
TIIOMAS
L. 1,15ix’1‘%
B. Benes~ow, and C. YACOOB. 1974. Characterization of a brain that maintains ChE activity of newt muscles cultured irk TlifYo. Fed. Pmr. 33: 622A. 19. SATTIN, ,4., and T. W. RAU. 1967. The effect of brain extracts on the accumulation of cyclic 3’,5’-AMP (CA) in slices of guinea pig (GP) cerebral cortex. Fed. I’YOC. 26: 707A. 20. SI~GINS, G. R., E. F. BATTENBERC;, B. J. HOFFER, and F. E. BLOOM. 1973. Noradrenergic stimulation of cyclic adenosine monophosphate in rat Purkinje neurons : An immunocytochen~ical study. .Scicwr 179 : 585-588.
XECROLOGY
49, 716-724 (1975)
Effect of Denervation
on Muscle Cyclic AMP in the Newt THOMAS
Section
of
Cytology, Yale New Haven, Received
L. LENTZ~ University Corm&cut June
School 06510
of
Medicine,
21, 1975
The effects of denervation ir, z&o and of nerve extracts in vitro on muscle cyclic AMP levels were measured to determine whether nerves influence muscle cyclic AMP. Denervation in viz10 resulted in an initial small decrease followed by a large increase in muscle cyclic AMP. Cyclic AMP levels declined as the muscle became reinnervated. After brief times in culture (l-3 days), cyclic AMP levels were lower in untreated muscles than in those cultured in the presmence of nerve extracts. At longer times, cyclic AMP was the same or higher in untreated muscles.
INTRODUCTION In addition to the short-term changes associated with transmission of the nerve impulse, the motor neuron exerts several long-term regulatory influences, defined as “trophic,” on the molecular specializations of the skeletal muscle fiber. One such influence is the neural regulation of muscle cholinesterase (ChE) activity, which is induced in developing or regenerating muscle by the nerve fiber and is reduced following denervation (3, 6-8, 11). The sensitivity of muscle ChE to neural influences has been utilized in an ill vitro system to assay potential trophic factors (9, 13, 17). The assay tests the ability of substances or experimental treatments to prevent the decreasein ChE activity of cultured skeletal muscle occurring as a result of denervation. Utilizing this system, it has been shown that nerve explants and extracts are effective in maintaining ChE activity of cultured muscle. Furthermore, adenosine 3’S’-cyclic monophosphate (cyclic AMP) in combination with theophylline, an inhibitor of cyclic nucleotide phospho1 Supported by a grant (GB-20902) from the National Science Foundation. I would like to thank Dr. James Nathanson, Ms. Linda Brown, and Ms. Janice Chester for assistance in the experimental procedures and Dr. Paul Greengard for reading the manuscript. 716 Copyright 0 1975 by Academic Press, Inc. All rights of repwduction in any form reserved.
CYCLIC
AMP
717
diesterase, and dibutyryl cyclic AMP have been found to have an effect similar to that of nerve explants in maintaining the ChE activity of denervated muscle in organ culture (IO, 1::7 IS). In addition, cyclic AMP has been implicated in another trophically regulated system, the regenerating limb of the newt, where it stimulates mitotic activity and regenerative growth (4). The action of cyclic A?IIP in mediating the effects of polypeptide hormones is well known. In addition, evidence has been presented recently that the actions of some neurotransmitters are mediated by cyclic AMP (5, 20). It has been hypothesized by McVahon (15) that cyclic nucleotides, under the regulatory influence of inducers which may include neurotransmitters, are involved in the regulation of developmental processes throughout the eukaryotes. In view of evidence that in certain situations neurons can exert postsynaptic effects on cyclic AMP levels, and the observation that cyclic AMP mimics the effect of nerve on muscle ChE, it seems possible that the trophic effects of nerve on muscle might be mediated by cyclic AMP formed in response to the trophic influence (10). If such trophic effects are mediated by cyclic AMP, the motor neuron should exert regulatory influences on muscle adenylate cyclase or possibly phosphodiesterase activities resulting in alterations of muscle cyclic AMP levels. The present study was carried out to measure the effects of denervation in zlizfo and of nerve extracts irr zifvo on cyclic AMP levels of muscle to determine whether innervation influences muscle cyclic AMI’. MATERIALS
AKD
METHODS
These studies were performed on the newt Trifunrs zh2escens. Cyclic AMP was measured in muscles of normal animals, after denervation, after tenotomy, after elimination of the blood supply to the limb, and in muscles placed in organ culture and treated with nerve extracts. In all cases,cyclic AMP was measured in paired forelimb muscles, one of which was treated experimentally, while the other served as an untreated control. Five to 1S pairs of muscle were used in each experiment. Operative procedures were performed on newts anesthetized in O.Zc/u chloretone. Denervation was achieved in one forelimb by exposing the hrachial plexus and remoGng a 1 mm segment of the brachial nerve cords. This procedure eliminates the motor and sensory supply (nerves 3, 4, and 5) to the limb. A sham operation was performed on the opposite limb, and the nerves left intact. The triceps muscles from the denervated limb and the innervated opposite limb were removed at various intervals for assay of cyclic ;\nrlj content. At various times. the brnchial plexus and triceps muscles of tlcnervatetl animals \vere exposed and examined
718
THOMAS
L.
LENT2
under a dissecting microscope to follow the time course of reinnervation of the limb. Tenotomy was accomplished by cutting the tendinous insertions and origins of the triceps muscle. The blood supply to the limb was eliminated by pinching and severing the brachial vessels. Muscles were cultured as described previously (9). One muscle of each pair was cultured in media containing newt brain extract prepared as previously described (13). Concentrations of extract known to maintain cholinesterase activity were used. The effect of liver extract on cyclic AMP was also tested. For cyclic AMP assay, muscles were removed and homogenized with a glass homogenizer in 1 ml cold 985% ethanol-0.2N HCl. If the muscles were small, two or three were pooled. The homogenate was centrifuged at 9,000 rpm in an International bench model centrifuge. Two 0.3 ml aliquots of each sample were removed and dried for cyclic AMP assay. The precipitate was assayed for protein with the method of Lowry et al. (14). The amount of cyclic AMP in each aliquot was measured by the method of Brown, Ekins, and Albano (1). Cyclic AMP content was calculated from a linear standard curve constructed with from 0.1 to 1.4 pmole of cyclic AMP. The amount of cyclic AMP per muscle was determined and divided by the protein content of each muscle to give pmole cyclic AMP per milligram of protein, RESULTS Individual newts differ in muscle cyclic AMP content; however, cyclic AMP content of triceps muscles from the same animal is the same (Table 1) . Thus, by utilizing pairs of muscles from the same animal, the samples are paired or nonindependent and the differences between members of pairs can be treated as single observations. In Tables 1 and 2, mean values for cyclic AMP levels are shown for control muscles, experimental muscles, and the differences between the two. The standard error of the mean (SEM) is given for the differences which are tested by the t distribution at the 0.05 or 0.01 significance levels. Cyclic AMP levels of denervated muscle undergo a relatively small, but significant decline in the first 2 days after nerve transection (Table 1, Fig. 1) . Thereafter, cyclic AMP rises to levels considerably higher than in the normally innervated muscles of the same animals reaching a maximum at 3 to 4 weeks. At this time, the denervated muscles are grossly smaller and atrophied. Cyclic AMP levels then begin to decline as the muscles are reinnervated, enlarge, and resume function. In tenotomized muscles, cyclic AMP undergoes a rapid and large increase. A transient decrease such as occurs following denervation did not occur after tenotomy. Cutting
Blood
muscles
vessels
Tenotomy
Denervation
Normal
Procedure
cut
Id
Id 16d
lh 4h Id 2d 3d 4d 8d 16d 24d 49d
Time
6
6 7
14 12 16 7 12 11 14 9 6 7
13
Number pairs
of
EFFECT
TABLE
1.50
Intact
2.07 2.65
Intact
1.99 2.83 3.17 2.78 2.03 3.27 1.92 2.33 1.68 4.00
Sham
3.64
1
3.62
Left
OS MUSCLE
1.53
cut
2.34 6.70
cut
1.90 2.61 2.32 2.07 1.83 3.52 1.93 2.84 3.81 6.09
Denervated
(pmol/mg protein)
Right
CAMP
OF DENERVATION
0.02
0.03
0.27 4.05
f
f f
f f f f f f f f f zk
f
0.07
0.12 0.33
0.21 0.25 0.33 0.28 0.16 0.37 0.21 0.24 0.19 0.70
0.35
of differences +SEM
-0.09 -0.23 -0.85 -0.71 -0.21 -0.26 0.01 0.51 2.13 2.09
Mean
CAMP
>o.os
>o.os
>o.os >o.os <0.0.5
>o.os
P
+2
+13 +153
+127 +59
+22
-8 -27 -26 -10 -7 +1
-5
-1
Difference G)
5
w
720 the blood vessels to the 1iIlllJ (lit1 not affect cyclic i\lll’ Icvels at 1 day. Thus, the changes in cJ.clic :jJl I’. at least at this time, are not the result of possible interference of the blootl supply by the (Jpxdve procedures. At longer times after blood vessel section, the muscles became necrotic so that it was not possible to measure cyclic L1hlP. The differences between untreated nluscles and muscles exposed to nerve extract ilz. z&o followed a l)attern similar to the differences between denervated and innervated mukcles ill zko during the first 3 days (Table 3). After 1 to 3 days in culture, cyclic AMP levels in untreated (comparable to denervated) muscles were lower than in those cultured in the presence of nerve estracts, although it appears that cyclic AMP decreases in both treated and untreated muscle in culture. After 8 days in culture, cyclic AMP was the same or greater in the untreated mu4e than in the treated muscle. Liver extract had no effect on muscle cyclic AMP. DISCUSSION It was found in the newt that loss of innervation results in an initial small decrease followed by a large and prolonged rise in muscle cyclic AMP levels. These findings indicate the motor nerve fiber may exert some control on the cyclic AMP levels of skeletal muscle. The significance of the observed changes in relation to trophic mechanisms is difficult to determine, however. The initial decrease in cyclic AMP in denervated muscle is consistent with the hypothesis that trophic effects might be mediated by cyclic AMP formed in response to the neuronal influence. In addition, cyclic AMP levels ia z&o could be maintained for 1 to 3 days by brain extracts. Brain extracts have also been found to stimulate accumulation of cyclic AMP in slices of cerebral cortex after very brief exposure (19). On the other hand, the large subsequent increase in cyclic AMP levels in denervated muscle as well as the lower levels in muscle treated with nerve extracts in longer term cultures, seem to indicate innervation might normally suppress cyclic AMP levels. Carlsen (2)) in studying cyclic AMP changes in denervated rat gastrocnemius, found that cyclic AMP levels increased following denervation and were reduced with reinnervation. These changes were similar to those described in the present study, although an initial increase in cyclic AMP was not observed and the changes occurred much more rapidly in the rat than in the newt. Carlsen (2) found in addition that the onset of the postdenervation increase in cyclic AMP depended on the length of the nerve stump. He suggested that muscle activity might suppress cyclic AMP, but that, in addition, a chemical factor supplied by the nerve regulates the cyclic AMP system.
Brain Brain Brain Brain Brain Liver
Extract
(0.07 (0.31 (0.07 (0.31 (0.31 (0.07
mg/ml) mg/ml) mg/mI) mg/ml) mg,/ml) mg/ml)
(mg protein,‘ml medium)
__-
1 1 3 3 8 3
Time (days)
6 12 18 12 12 5
Number pairs
of
NERVETISSVE
CAMP
1.88 2.14 0.85 0.84 0.63 1.86
Plain medium
-.
TABLE
2
2.25 2.36 1.68 1.10 0.54 1.93
Medium fextract
(pmol,‘mg protein)
EXTRACT-EFFECTOS
* Lt f i * zk
0.15 0.05 0.19 0.14 0.24 0.56
of differences GEM
CAMP
0.37 0.22 0.83 0.27 -0.09 0.07
Mean
MUSCLE
_---.-
P-o.05
P
+20 +10 +97 +32 -15 +4
Difference (yy) si p. c c, > R 9
722
TIIOMAS
L.
LENT2
-so
FIG. 1. Effect of denervation and tenotomy on cyclic AMP levels in the triceps muscle of the newt. Denervation of muscle results in an initial decrease followed by a large increase in muscle cyclic AMP. Tenotomy produces a large increase in cyclic AMP. See Table 1 for data and statistics.
Caution is required, however, in drawing conclusions regarding neural influences on cyclic AMP solely on the basis of cyclic AMP measurements, since there can be increased turnover of cyclic AMP and a physiological effect without accumulation or even with a decline in total cyclic AMP during a hormonally regulated process. In addition, it is probable that a number of factors influence cyclic AMP levels of muscle, and these effects must be distinguished from those of nerve. The effects of tenotomy, for example, might indicate that muscle activity has an influence on cyclic AMP, acting to suppress it. The large increase in cyclic AMP in denervated muscle could, therefore, be the result of muscle inactivity following denervation. It should be noted, however, that in the cat, tenotomy of a single muscle results in little or no change in electromyographic activity (16). It appears at this point that denervation results in alterations in muscle cyclic AMP levels and that cyclic AMP plays a role in the maintenance of at least one of the differentiated characteristics (ChE) of the muscle fiber which is also dependent on innervation. The relation between innervation and cyclic AMP and the mechanism of action of cyclic AMP
remain unclear. Further information on the action of nerves in regulation of muscle cyclic AMP could be obtained by determining the effect of neural inAuences on muscle adenyl cyclase, phosphodiesterase, and protein kinases and their substrates.
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724 1X. RATHBOPITE, M. P., factor from chick
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