EXPERIMENTAL
NEUROLOGY
Denervated
Frog
53, 64-70 (1976)
Skeletal Kinetics
Muscle: Calcium of Exchange
Content
and
JOHN R. PICKEN AND ALBERT C. KIRBY 1 Departlrlcrrt
of Physiology,
Sclzool of Medicine, Cleveland, Ohio Received
April
Case Western
Resrrve
University,
44106 7, 1976
Calciumcontent and exchangein denervatedfrog extensor longus digitorum (ELD IV) muscles were examined using the efflux technique. Time constants of exchange were found to be similar in both intact and denervated muscle. Calcium content of denervated muscle was significantly greater than
that of intact. Although four componentsof exchangecould be determined, the increase in calcium content was largely restricted to components believed to be extracellular space and sarcoplasmic reticulum. The data are discussed in relation to morpho!ogical and radioautographic findings from other laboratories.
INTRODUCTION The crucial role of calcium in skeletal muscle contraction is well understood and widely accepted (3, 19). It might be expected that functional changes in muscle could be a reflection of an alteration in intracellular calcium compartmentalization and metabolism. Specifically, work from our laboratory has demonstrated a dependence of denervated muscle contractile response (9, 17, 18) on extracellular calcium. These initial observations led us to undertake a more detailed investigation of calcium content and exchange in denervated frog skeletal muscle. Calcium exchange in denervated mammalian muscle has been studied (8). Intracellular calcium distribution has been studied by us (10) and numerous others (2, 4, 15, 16, 20, 21). The net result of these previous studies is that muscle calcium is found distributed among several coml This work was supportedby National Institutes of Health Grants NS-10196and GM-00899.J. R. Picken held an American Heart Association,NortheastOhio Chapter, postdoctoral fellowship during a portion of this work. Portions of this paper form part of a thesis submitted by John R. Picken in partial fulfillment of the requirements for the degree of Doctor of Philosophy. We thank Dr. Barry D. Lindley for his advice, criticism, and encouragement of both the experiments and the manuscript. 64 Copyright All rights
1976 by Academic CI? reproduction in any
CALCIUM
IN
DEh‘ERVATED
MUSCLE
6.5
partments and exchanges with time constants ranging from a few minutes to several hours. The present study was undertaken in an attempt to determine if muscle calcium content changes after denervation and to further determine the intracellular location of any calcium changes that might occur. We will present evidence in this paper showing that intracellular calcium content increases after denervation of skeletal muscle and that this increased amount of calcium appears to be associated with the sarcoplasmic reticulun~. Portions of this work have been reported previously ( 14). METHODS Detailed methods of care, handling of the frogs (9) and of isotope experiments (10) have been reported previously. An outline of the procedure is given. The frogs were denervatecl by transection of the sciatic nerve at the iliococcygeal junction, then maintained at room temperature until the day of the experiment. At this time both the denervated extensor longus digitorum (ELD IV) muscle and its contralateral innervated control (referred to as intact hereafter) were dissected out, equilibrated in Ringer’s solution containing @Ca2+ and then washed in separate but identical efflux chambers with fresh Ringer’s after isotopic equilibration. Samples were collected at set intervals and counted on a scintillation counter. To determine the sequential change in calciutll content after denervation, a modification of this approach was used. After the time constants of the components were determined in preliminary experiments, it became possible to determine the calcium contents of the appropriate components by a less complicated procedure. In the case of the fastest component (7 = 2.7 min) the muscles were exposed to 45Ca2+ for 6 min, then washed 1 min in Ringer’s solution to clear the extracellular space and digested in Nitric acid. Ca2+ content of this component could be calculated using two corrections: First, calcium content of the component had not reached specific activity equilibration during the influx period and second, some calcium was lost during the washout period. Compensation for the two factors using an average time constant value allowed calculation of calcium content. A similar procedure was used for the slowest component (T = 1026 min) except that the influx period was 3 hr and the washout, 90 min. Total calcium was analyzed by ashing muscles and measuring against standards on an atomic absorption spectrophotometer; full details are in Kirby, Lindley, and Picken (10). RESULTS 45Ca”+ R’askout in Intact and Denevaated Mzlscle. Figure 1 is a desaturation plot of c5Ca2+ content in both intact and denervated frog skeletal muscle after the first 3 min of exchange. The elevated %a?+ content of
66
PICKEN
AND
KIRBY
FIG. 1. Desaturation plot of “Ca’+ content of denervated (A) and intact ( l ) ELD IV muscles. Points are experimentally obtained, the lines are calculated.
the denervated muscle is apparent although the kinetics of exchange appear similar from this figure. Basically, the method is as follows: The points beyond 150 min after start of the washout are fitted to straight lines. The slopes of these lines reflect the time constants and the intercepts. The calcium contents of those particular components of washout can be calculated by utilizing appropriate corrections. This linear component is then subtracted from the points earlier in time, revealing additional components. This procedure can be continued until the washout data can be broken down into a series of exponentials. Detailed procedures of data analysis and theoretical consideration of the problems in compartmental analysis were published previously (10). Three components of exchange can be determined from these experiments. Time constants and component sizes are tabulated in Table 1, using data obtained from nine matched pairs of intact and denervated ELD IV muscles. Exchange data on intact muscles have been published (10) previously. The exchange time constants are the same in both sets of muscles but the estimated Ca”+ contents of the fastest and slowest washout components are significantly greater in the denervated muscle (mean length of denervation = 35 days). The calcium content of the intermediate component is elevated as well but not significantly. Extracellztlar Space Calcium Content and Exchange. A large amount of calcium was found to exchange with a time constant of 26 set, a figure compatible with washout of a cylinder the size of the ELD IV muscle (10). The amount of calcium in this component was calculated to be 1.78 mlvr/kg in denervated muscle and 1.13 mM/kg in intact muscle, an amount of Ca2+ significantly greater than can be accounted for in the aqueous extracellular compartment assuming an extracellular space of 20% tissue wet weight (13). This extra calcium most likely represents surface membrane, t-tubular, and connective tissue-bound calcium that is in rapid equilibrium
CALCIUM
IN
DENERVATED
TABLE Calcium
67
MUSCLE
1
Exchange after First Intact and Denervated
3 Min of Washout Toe Muscles
for
Intact Time
constant
Pool size (mmole/kg)
(min)
Comp Comp Comp
1 2 3
2.7 zk 0.2c 32 k 4.0 1244 zt 85.0
Flux (pmole/cm2
0.20 f 0.02 0.09 f 0.01 0.54 zk 0.06 Total
set)
1.27 0.048 0.0076
= 0.83
Denervated” Camp Comp Camp
1 2 3
0.35 f o.os* 0.15 f 0.03 0.94 f 0.09**
2.5 It 0.2 39 * 2.0 1026 zt 70.0 Total
2.35 0.067 0.0166
= 1.44
n Means f SE of mean for nine experiments. 6 Mean denervation time was 35 days. *r < 0.025. ** I’ < 0.001.
with the extracellular space. The calcium content of this component is significantly elevated after denervation. Total Calcium. Total calcium determined by atomic absorption spectrophotometry in denervated muscle was 3.22 mmole/kg. The sum of extracellular-space calcium and the intracellular exchangeable components was 2.95 mmole/kg, with the difference being less than 10% of the total. These data are tabulated in comparison with intact muscle in Table 2. Thus, we feel that in denervated muscle, as in intact, all the muscle calcium is exchangeable on a time scale of hours. TABLE Calcium ___-
Content
2
of Frog
Toe
Intact
(nmole/kg) Extracellular Intracellular Total exchangeable TotaP Difference d Atomic
Muscle
-~-_
absorption
spcctrophotomctry.
1.13 0.83 1.96 2.00 -0.04
Denervated (nmole/kg) 1.78 1.44 3.22 2.95 $0.27
68
PICKEN
1 0
, , / 2
4
6
AND
KIRBY
, , , , , , , , 8
10 DAYS
12
14
16
18
20
22
24
I I I 26
28
30
32
POST-DENERVATJON
FIG. 2. A-Ratio of calcium content of the fastest intracellular compartment (T = 2.5, 2.7 mm), of denervated muscle divided by that of intact muscle, as a function of time after denervation. The shaded bar represents the approximate time of neuromuscular junction failure. B-Ratio of calcium content of the slowest intracellular compartment (7 = 1026, 1244 min), of denervated muscle divided by that of intact muscle, as a function of time after denervation. The solid bar represents the approximate time of neuromuscular junction failure.
Sequence of Changes after Denervation. Because the calcium contents of the fastest and slowest intracellular components were significantly elevated following denervation, we felt it important to analyze the changes in these components as a function of time after denervation. Figure 2A shows that the calcium content of the fastest exchanging intracellular component (T = 2.7 min) is elevated immediately after denervation and continues to rise. In contrast, the calcium content of the most slowly exchanging component remains unchanged and may even fall in the first week after denervation but begins to rise about the time of failure of the neuromuscular junction (6 to 8 days), Fig. 2B. DISCUSSION In denervated frog skeletal muscle, as in intact, we have demonstrated that calcium exchange can be described as the sum of four exponentials.
CALCIUM
IN
DENERVATED
MUSCLE
69
Three of these components we believe are intracellular. In addition, we have shown that virtually all of the cell calcium is exchangeable. Many aspects of muscle function appear to be related to the nerve supply (5) ; included among these are metabolism, structure, and contractility. Therefore, it does not seem surprising that muscle calcium content should vary as a consequence of denervation. Hines and Knowlton (6) discovered that the muscle calcium content of rat gastrocnemius muscle approximately doubled by 28 days after denervation. They showed that a small portion of this extra calcium was associated with connective tissue but were not able to offer an explanation for the bulk of the calcium increase. Isaacson and Sandow (8) have shown that calcium flux is elevated in denervated rat extensor digitorum longus and that greater c5CaZ+ flux occurs in caffeine contracture of the denervated muscle. Howell, Fairhurst, and Jenden (7) have shown that Ca?+ accumulating ability of muscle is enhanced after denervation. Brody ( 1) demonstrated that calcium accumulating ability of muscle microsomes is enhanced after denervation. Therefore, we feel that the functional significance of the increased calcium content of the fastest and slowest exchanging intracellular components in denervation may be related to sarcoplasmic reticulum function. We have proposed a model of calcium localization and exchange (10) based on work of Winegrad (20, 21). Our previously reported model predicts that the fastest exchanging intracellular component is terminal cisternae calcium and the slowest, longitudinal reticulum calcium. Muscatello, Margreth, and Aloisi (12) have shown that among the consequences of denervation in frog skeletal muscle is an enlargement of the sarcoplasmic reticulum, first in the area of t-tubules and terminal cisternae, later in the longitudinal reticulum. We feel that the time sequence of changes in calcium content when compared to the morphological observations discussed above provide further support for this hypothesis. Kirby, Lindley, and Picken (9) and Lindley et al. (11) have published evidence that the earliest contractile change seen as a consequence of denervation is a profound effect of extracellular calcium concentration on potassium contractures. Stuesse, Lindley, and Kirby (1s) showed that the underlying basis for this response was a much faster rate of relaxation of the potassium contracture of denervated frog muscle. Among the changes which could cause this result is an increased rate of uptake of free calcium during the contracture. This hypothesis seems consistent with both the observed expansion of the sarcoplasmic reticulum following denervation and the greater calcium content of the reticulum. REFERENCES 1.
I. A. 1966. Relaxing factor in denervated muscle: A possible explanation for fibrillations. A~zcr. J. Physiol. 211 : 1277-1280.
BRODY,
70
PICKEN
AND
KIRBY
2. CURTIS, B. A. 1970. Calcium et&x from frog twitch muscle fibers. I. Gen. Physiol. 55 : 243-253.
EBASHI, 5, and M. ENDO. 1968. Calcium ion and muscle contraction. Prog. Biophys. Mol. Biol. 18: 123-183. 4. GILBERT, D. L., and W. 0. FENN. 1957. Calcium equilibrium in muscle. J. Gcn. 3.
Physiol. 5.
40 : 393-408.
GUTH, L. 1968. “Trophic”
influences of nerve on muscle. Physiol.
Rev.
48:
645-
687.
HINES, H. M., and G. C. KNOWLTON. 1933. Changes in the skeletal muscle of the rat following denervation. Amer. J. Physiol. 104: 379-391. 7. HOWELL, J. N., A. S. FAIRHURST, and P. J. JENDEN. 1966. Alterations of the calcium accumulating ability of striated muscle following denervation. Life Sci. 5 : 439446. 8. ISAACSON, A., and A. SANDOW. 1967. Caffeine effects on radiocalcium movement in normal and denervated rat skeletal muscle. J. Pharmac. Exp. Ther. 155 : 3766.
388. 9.
10. 11. 12. 13.
14. 15. 16.
KIRBY, A. C., B. D. LINDLEY, and J. R. PICKEN. 1973. Calcium dependence of potassium contractures in denervated frog muscle. Amer. J. Physiol. 225: 166170. KIRBY, A. C., B. D. LINDLEY, and J. R. PICKEN. 1975. Calcium content and exchange in frog skeletal muscle. J. Physiol. 253: 37-52. LINDLEY, B. D., A. C. KIRBY, S. C. STEUSSE, and J. R. PICKEN. 1973. Mechanical threshold and inactivation in denervated frog muscle. Amer. J. Physiol. 225: 171-176. MUSCATELLO, V., A. MARGRETH, and M. ALOISI. 1965. On the differential response of sarcoplasm and myoplasm to denervation in frog muscle. J. Cell. Viol. 27: l-24. PICKEN, J. R. 1974. Cal&m contelrt and excharzge in intact alzcl dertervated skeletal nzuscle. Ph.D. dissertation, Case Western Reserve University, Cleveland, Ohio. PICKEN, J. R., A. C. KIRBY, and B. D. LIN~LEY. 1974. ‘“Ca” efflux from intact and denervated frog skeletal muscle. Fed. Proc. 33: 383. SHANES, A. M., and C. P. BIANCHI. 1959. The distribution and kinetics of release of radiocalcium in tendon and skeletal muscle. J. Gen. Physiol. 42 : 1123-1137. SHANES, A. M., and C. P. BIANCHI. 1959. Radiocalcium release by stimulated and potassium treated sartorius muscles of the frog. J. Gen. Physiol. 43: 481493.
17. STUESSE, S. C., and B. D. LIN~LEY. 1975. Contractile inactivation in frog single denervated muscle fibers. Amer. J. Physiol. 229: 1492-1497. 18. STUESSE, S. C., B. D. LINDLEY, and A. C. KIRBY. 1974. Potassium contractures of frog single denervated muscle fibers: Time course and central spread. Amer. J. Physiol.
227 : 200-208.
19. WEBER, A., and J. M. MURRAY. 1973. Molecular control mechanisms in muscle contraction. Physiol. Rev. 53 : 612-673. 20. WINEGRAD, S. 1968. Intracellular calcium movements of frog skeletal muscle during recovery from tetanus. J. Gen. Physiol. 51: 65-83. 21. WINEGRAD, S. 1970. The intracellular site of calcium activation of contraction in frog skeletal muscle. J. Gen. Physiol. 55: 77-88.
NEUROLOGY
Denervated
Frog
53, 64-70 (1976)
Skeletal Kinetics
Muscle: Calcium of Exchange
Content
and
JOHN R. PICKEN AND ALBERT C. KIRBY 1 Departlrlcrrt
of Physiology,
Sclzool of Medicine, Cleveland, Ohio Received
April
Case Western
Resrrve
University,
44106 7, 1976
Calciumcontent and exchangein denervatedfrog extensor longus digitorum (ELD IV) muscles were examined using the efflux technique. Time constants of exchange were found to be similar in both intact and denervated muscle. Calcium content of denervated muscle was significantly greater than
that of intact. Although four componentsof exchangecould be determined, the increase in calcium content was largely restricted to components believed to be extracellular space and sarcoplasmic reticulum. The data are discussed in relation to morpho!ogical and radioautographic findings from other laboratories.
INTRODUCTION The crucial role of calcium in skeletal muscle contraction is well understood and widely accepted (3, 19). It might be expected that functional changes in muscle could be a reflection of an alteration in intracellular calcium compartmentalization and metabolism. Specifically, work from our laboratory has demonstrated a dependence of denervated muscle contractile response (9, 17, 18) on extracellular calcium. These initial observations led us to undertake a more detailed investigation of calcium content and exchange in denervated frog skeletal muscle. Calcium exchange in denervated mammalian muscle has been studied (8). Intracellular calcium distribution has been studied by us (10) and numerous others (2, 4, 15, 16, 20, 21). The net result of these previous studies is that muscle calcium is found distributed among several coml This work was supportedby National Institutes of Health Grants NS-10196and GM-00899.J. R. Picken held an American Heart Association,NortheastOhio Chapter, postdoctoral fellowship during a portion of this work. Portions of this paper form part of a thesis submitted by John R. Picken in partial fulfillment of the requirements for the degree of Doctor of Philosophy. We thank Dr. Barry D. Lindley for his advice, criticism, and encouragement of both the experiments and the manuscript. 64 Copyright All rights
1976 by Academic CI? reproduction in any
CALCIUM
IN
DEh‘ERVATED
MUSCLE
6.5
partments and exchanges with time constants ranging from a few minutes to several hours. The present study was undertaken in an attempt to determine if muscle calcium content changes after denervation and to further determine the intracellular location of any calcium changes that might occur. We will present evidence in this paper showing that intracellular calcium content increases after denervation of skeletal muscle and that this increased amount of calcium appears to be associated with the sarcoplasmic reticulun~. Portions of this work have been reported previously ( 14). METHODS Detailed methods of care, handling of the frogs (9) and of isotope experiments (10) have been reported previously. An outline of the procedure is given. The frogs were denervatecl by transection of the sciatic nerve at the iliococcygeal junction, then maintained at room temperature until the day of the experiment. At this time both the denervated extensor longus digitorum (ELD IV) muscle and its contralateral innervated control (referred to as intact hereafter) were dissected out, equilibrated in Ringer’s solution containing @Ca2+ and then washed in separate but identical efflux chambers with fresh Ringer’s after isotopic equilibration. Samples were collected at set intervals and counted on a scintillation counter. To determine the sequential change in calciutll content after denervation, a modification of this approach was used. After the time constants of the components were determined in preliminary experiments, it became possible to determine the calcium contents of the appropriate components by a less complicated procedure. In the case of the fastest component (7 = 2.7 min) the muscles were exposed to 45Ca2+ for 6 min, then washed 1 min in Ringer’s solution to clear the extracellular space and digested in Nitric acid. Ca2+ content of this component could be calculated using two corrections: First, calcium content of the component had not reached specific activity equilibration during the influx period and second, some calcium was lost during the washout period. Compensation for the two factors using an average time constant value allowed calculation of calcium content. A similar procedure was used for the slowest component (T = 1026 min) except that the influx period was 3 hr and the washout, 90 min. Total calcium was analyzed by ashing muscles and measuring against standards on an atomic absorption spectrophotometer; full details are in Kirby, Lindley, and Picken (10). RESULTS 45Ca”+ R’askout in Intact and Denevaated Mzlscle. Figure 1 is a desaturation plot of c5Ca2+ content in both intact and denervated frog skeletal muscle after the first 3 min of exchange. The elevated %a?+ content of
66
PICKEN
AND
KIRBY
FIG. 1. Desaturation plot of “Ca’+ content of denervated (A) and intact ( l ) ELD IV muscles. Points are experimentally obtained, the lines are calculated.
the denervated muscle is apparent although the kinetics of exchange appear similar from this figure. Basically, the method is as follows: The points beyond 150 min after start of the washout are fitted to straight lines. The slopes of these lines reflect the time constants and the intercepts. The calcium contents of those particular components of washout can be calculated by utilizing appropriate corrections. This linear component is then subtracted from the points earlier in time, revealing additional components. This procedure can be continued until the washout data can be broken down into a series of exponentials. Detailed procedures of data analysis and theoretical consideration of the problems in compartmental analysis were published previously (10). Three components of exchange can be determined from these experiments. Time constants and component sizes are tabulated in Table 1, using data obtained from nine matched pairs of intact and denervated ELD IV muscles. Exchange data on intact muscles have been published (10) previously. The exchange time constants are the same in both sets of muscles but the estimated Ca”+ contents of the fastest and slowest washout components are significantly greater in the denervated muscle (mean length of denervation = 35 days). The calcium content of the intermediate component is elevated as well but not significantly. Extracellztlar Space Calcium Content and Exchange. A large amount of calcium was found to exchange with a time constant of 26 set, a figure compatible with washout of a cylinder the size of the ELD IV muscle (10). The amount of calcium in this component was calculated to be 1.78 mlvr/kg in denervated muscle and 1.13 mM/kg in intact muscle, an amount of Ca2+ significantly greater than can be accounted for in the aqueous extracellular compartment assuming an extracellular space of 20% tissue wet weight (13). This extra calcium most likely represents surface membrane, t-tubular, and connective tissue-bound calcium that is in rapid equilibrium
CALCIUM
IN
DENERVATED
TABLE Calcium
67
MUSCLE
1
Exchange after First Intact and Denervated
3 Min of Washout Toe Muscles
for
Intact Time
constant
Pool size (mmole/kg)
(min)
Comp Comp Comp
1 2 3
2.7 zk 0.2c 32 k 4.0 1244 zt 85.0
Flux (pmole/cm2
0.20 f 0.02 0.09 f 0.01 0.54 zk 0.06 Total
set)
1.27 0.048 0.0076
= 0.83
Denervated” Camp Comp Camp
1 2 3
0.35 f o.os* 0.15 f 0.03 0.94 f 0.09**
2.5 It 0.2 39 * 2.0 1026 zt 70.0 Total
2.35 0.067 0.0166
= 1.44
n Means f SE of mean for nine experiments. 6 Mean denervation time was 35 days. *r < 0.025. ** I’ < 0.001.
with the extracellular space. The calcium content of this component is significantly elevated after denervation. Total Calcium. Total calcium determined by atomic absorption spectrophotometry in denervated muscle was 3.22 mmole/kg. The sum of extracellular-space calcium and the intracellular exchangeable components was 2.95 mmole/kg, with the difference being less than 10% of the total. These data are tabulated in comparison with intact muscle in Table 2. Thus, we feel that in denervated muscle, as in intact, all the muscle calcium is exchangeable on a time scale of hours. TABLE Calcium ___-
Content
2
of Frog
Toe
Intact
(nmole/kg) Extracellular Intracellular Total exchangeable TotaP Difference d Atomic
Muscle
-~-_
absorption
spcctrophotomctry.
1.13 0.83 1.96 2.00 -0.04
Denervated (nmole/kg) 1.78 1.44 3.22 2.95 $0.27
68
PICKEN
1 0
, , / 2
4
6
AND
KIRBY
, , , , , , , , 8
10 DAYS
12
14
16
18
20
22
24
I I I 26
28
30
32
POST-DENERVATJON
FIG. 2. A-Ratio of calcium content of the fastest intracellular compartment (T = 2.5, 2.7 mm), of denervated muscle divided by that of intact muscle, as a function of time after denervation. The shaded bar represents the approximate time of neuromuscular junction failure. B-Ratio of calcium content of the slowest intracellular compartment (7 = 1026, 1244 min), of denervated muscle divided by that of intact muscle, as a function of time after denervation. The solid bar represents the approximate time of neuromuscular junction failure.
Sequence of Changes after Denervation. Because the calcium contents of the fastest and slowest intracellular components were significantly elevated following denervation, we felt it important to analyze the changes in these components as a function of time after denervation. Figure 2A shows that the calcium content of the fastest exchanging intracellular component (T = 2.7 min) is elevated immediately after denervation and continues to rise. In contrast, the calcium content of the most slowly exchanging component remains unchanged and may even fall in the first week after denervation but begins to rise about the time of failure of the neuromuscular junction (6 to 8 days), Fig. 2B. DISCUSSION In denervated frog skeletal muscle, as in intact, we have demonstrated that calcium exchange can be described as the sum of four exponentials.
CALCIUM
IN
DENERVATED
MUSCLE
69
Three of these components we believe are intracellular. In addition, we have shown that virtually all of the cell calcium is exchangeable. Many aspects of muscle function appear to be related to the nerve supply (5) ; included among these are metabolism, structure, and contractility. Therefore, it does not seem surprising that muscle calcium content should vary as a consequence of denervation. Hines and Knowlton (6) discovered that the muscle calcium content of rat gastrocnemius muscle approximately doubled by 28 days after denervation. They showed that a small portion of this extra calcium was associated with connective tissue but were not able to offer an explanation for the bulk of the calcium increase. Isaacson and Sandow (8) have shown that calcium flux is elevated in denervated rat extensor digitorum longus and that greater c5CaZ+ flux occurs in caffeine contracture of the denervated muscle. Howell, Fairhurst, and Jenden (7) have shown that Ca?+ accumulating ability of muscle is enhanced after denervation. Brody ( 1) demonstrated that calcium accumulating ability of muscle microsomes is enhanced after denervation. Therefore, we feel that the functional significance of the increased calcium content of the fastest and slowest exchanging intracellular components in denervation may be related to sarcoplasmic reticulum function. We have proposed a model of calcium localization and exchange (10) based on work of Winegrad (20, 21). Our previously reported model predicts that the fastest exchanging intracellular component is terminal cisternae calcium and the slowest, longitudinal reticulum calcium. Muscatello, Margreth, and Aloisi (12) have shown that among the consequences of denervation in frog skeletal muscle is an enlargement of the sarcoplasmic reticulum, first in the area of t-tubules and terminal cisternae, later in the longitudinal reticulum. We feel that the time sequence of changes in calcium content when compared to the morphological observations discussed above provide further support for this hypothesis. Kirby, Lindley, and Picken (9) and Lindley et al. (11) have published evidence that the earliest contractile change seen as a consequence of denervation is a profound effect of extracellular calcium concentration on potassium contractures. Stuesse, Lindley, and Kirby (1s) showed that the underlying basis for this response was a much faster rate of relaxation of the potassium contracture of denervated frog muscle. Among the changes which could cause this result is an increased rate of uptake of free calcium during the contracture. This hypothesis seems consistent with both the observed expansion of the sarcoplasmic reticulum following denervation and the greater calcium content of the reticulum. REFERENCES 1.
I. A. 1966. Relaxing factor in denervated muscle: A possible explanation for fibrillations. A~zcr. J. Physiol. 211 : 1277-1280.
BRODY,
70
PICKEN
AND
KIRBY
2. CURTIS, B. A. 1970. Calcium et&x from frog twitch muscle fibers. I. Gen. Physiol. 55 : 243-253.
EBASHI, 5, and M. ENDO. 1968. Calcium ion and muscle contraction. Prog. Biophys. Mol. Biol. 18: 123-183. 4. GILBERT, D. L., and W. 0. FENN. 1957. Calcium equilibrium in muscle. J. Gcn. 3.
Physiol. 5.
40 : 393-408.
GUTH, L. 1968. “Trophic”
influences of nerve on muscle. Physiol.
Rev.
48:
645-
687.
HINES, H. M., and G. C. KNOWLTON. 1933. Changes in the skeletal muscle of the rat following denervation. Amer. J. Physiol. 104: 379-391. 7. HOWELL, J. N., A. S. FAIRHURST, and P. J. JENDEN. 1966. Alterations of the calcium accumulating ability of striated muscle following denervation. Life Sci. 5 : 439446. 8. ISAACSON, A., and A. SANDOW. 1967. Caffeine effects on radiocalcium movement in normal and denervated rat skeletal muscle. J. Pharmac. Exp. Ther. 155 : 3766.
388. 9.
10. 11. 12. 13.
14. 15. 16.
KIRBY, A. C., B. D. LINDLEY, and J. R. PICKEN. 1973. Calcium dependence of potassium contractures in denervated frog muscle. Amer. J. Physiol. 225: 166170. KIRBY, A. C., B. D. LINDLEY, and J. R. PICKEN. 1975. Calcium content and exchange in frog skeletal muscle. J. Physiol. 253: 37-52. LINDLEY, B. D., A. C. KIRBY, S. C. STEUSSE, and J. R. PICKEN. 1973. Mechanical threshold and inactivation in denervated frog muscle. Amer. J. Physiol. 225: 171-176. MUSCATELLO, V., A. MARGRETH, and M. ALOISI. 1965. On the differential response of sarcoplasm and myoplasm to denervation in frog muscle. J. Cell. Viol. 27: l-24. PICKEN, J. R. 1974. Cal&m contelrt and excharzge in intact alzcl dertervated skeletal nzuscle. Ph.D. dissertation, Case Western Reserve University, Cleveland, Ohio. PICKEN, J. R., A. C. KIRBY, and B. D. LIN~LEY. 1974. ‘“Ca” efflux from intact and denervated frog skeletal muscle. Fed. Proc. 33: 383. SHANES, A. M., and C. P. BIANCHI. 1959. The distribution and kinetics of release of radiocalcium in tendon and skeletal muscle. J. Gen. Physiol. 42 : 1123-1137. SHANES, A. M., and C. P. BIANCHI. 1959. Radiocalcium release by stimulated and potassium treated sartorius muscles of the frog. J. Gen. Physiol. 43: 481493.
17. STUESSE, S. C., and B. D. LIN~LEY. 1975. Contractile inactivation in frog single denervated muscle fibers. Amer. J. Physiol. 229: 1492-1497. 18. STUESSE, S. C., B. D. LINDLEY, and A. C. KIRBY. 1974. Potassium contractures of frog single denervated muscle fibers: Time course and central spread. Amer. J. Physiol.
227 : 200-208.
19. WEBER, A., and J. M. MURRAY. 1973. Molecular control mechanisms in muscle contraction. Physiol. Rev. 53 : 612-673. 20. WINEGRAD, S. 1968. Intracellular calcium movements of frog skeletal muscle during recovery from tetanus. J. Gen. Physiol. 51: 65-83. 21. WINEGRAD, S. 1970. The intracellular site of calcium activation of contraction in frog skeletal muscle. J. Gen. Physiol. 55: 77-88.
