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β-Adrenergic receptor-adenylate cyclase of denervated sarcolemmal membrane
EXPERIMENTAL
NEUROLOGY
59,
361-371
(1978)
P-Adrenergic Receptor-Adenylate Denervated Sarcolemmal P.
BLAISE
SMITH,
STUART
P.
GREFRATH,
Cyclase Membrane AND
STANLEY
of
H.
APPEL
l
Departme& of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27103, and Division of Neurology, Baylor College of Medicine, Houston. Texas 77025 Received
September
27,1977;
received December
revision
2,1977
Five days after motor denervation, mammalian skeletal muscle sarcolemma undergoes a 50% decline in basal and catecholamine-stimulated adenylate cyclase activity. Sodium fluoride- and S-guanylyl imidophosphate [ Gpp( NH) p) ]-stimulated activities also are depressed. However, properties of the /%adrenergic receptor in the plasma membrane preparation of denervated muscle are similar to those of normal innervated muscle. The number and affinity of specific (-) -[3H]dihydroalprenolol binding sites, the effects of catecholaminergic ligands on binding (isoproterenol > epinephrine > norepinephrine), the stimulatory effects of Gpp(NH)p on adenylate cyclase activity, and the shift in concentration of catecholamines required to activate adenylate cyclase in the presence of Gpp(NH)p were similar in normal and denervated membranes. Thus denervation appears to uncouple the adenylate cyclase response from p-adrenergic stimulation primarily by a loss of adenylate cyclase activity with no change in receptor properties.
INTRODUCTION Previous work from our laboratory characterized the ,8-adrenergic receptor-adenylate cyclase system in the sarcolemmal membrane isolated l This paper is dedicated to the memory of Dr. Stuart P. Grefrath, deceased. We thank Mrs. Tommye Campbell for expert secretarial assistance. This work was supported by Grant NS-07872 from the National Institutes of Health. Portions of this study were presented by Dr. P. Blaise Smith at the annual FASEB meeting, San Francisco, California, June 1976, and Dr. Stuart P. Grefrath at the annual meeting of the Society for Neuroscience, Toronto, Ontario, Canada, November 1976. Requests for reprints should be addressed to Dr. Appel at Baylor College of Medicine. Abbreviations : Gpp (NH) p--5’-guanylyl imidodiphosphate ; CAMP-adenosine 3’ : 5’-cyclic monophosphate. 361 0014-4886/78/0593-0361$02.00/O All
Copyright 0 1978 by Academic Press, Inc. rights of reproduction in any form reserved.
362
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GREFRATH,
AND
APPEL
from normal mammalian skeletal muscle (6, 7, 21). In these membranes adenylate cyclase activity was stimulated proportionately to the affinity of catecholamine hormone binding to the p-receptor. The kinetic properties of the enzyme and dihydroalprenoiol binding properties of the receptor suggested the presence of a coupled Ps-type adrenergic response similar to the physiological and pharmacological response of skeletal muscle to catecholamines. In the course of studies on the effect of denervation on the biochemical properties of the sarcolemma, it was found that catecholamine hormone stimulation of adenylate cyclase activity declined 50% after 5 days of denervation (22). This observation plus our characterization of the P-receptor, adenylate cyclase, and guanylyl nucleotide effect in normal muscle prompted the present investigation in which we examine each of these sarcolemmal components of the p-adrenergic response in denervated muscle. The object of this study was to determine whether loss of receptor or impairment of active enzyme was responsible for the diminution of catecholamine hormone-stimulated adenylate cyclase activity. The data suggest that denervation results in the loss of active enzyme and does not alter either receptor properties or guanylyl nucleotide regulation. METHODS
AND
MATERIALS
Denervation. Denervation was by excision of a 2-cm segment of the sciatic nerve from the midthigh region of female (200 to 250-g) Wistar rats. Sarcolemmal Membrane Isolation. An enriched fraction of the sarcolemma1 membrane was prepared from 5-day denervated muscle as previously described (1, 23). Briefly, rat lower limb muscle was minced and homogenized (Polytron, Brinkman Instrument Co.) in buffer consisting of 0.25 M sucrose, 0.2 mM Naa EDTA, and 0.2 mM Tris-HCl, pH 7.5. The homogenate was centrifuged 10 min at 1000 g and the pellet (nuclear fraction) retained for further hypertonic salt extractions. Crude membrane material resulting from this extraction procedure was subjected to continuous (15 to 35%) sucrose gradients and centrifugation was carried out at 200,000 g for 210 min. Membrane material distributing in the 19 to 24% zone of the gradient was removed, concentrated by centrifugation (48,000 g, 120 min) , and resuspended in 1 mM Tris-HCl buffer, pH 7.5, to a protein concentration of 2 to 4 mg/ml. All operations were carried out at 4°C. p-Receptor and adenylate cyclase studies were done immediately. This membrane preparation is characterized by possessing the highest specific and total activities of (i) Na+K+ (Mg*+) ATPase, (ii) sialic acid content, (iii) acetylcholine receptor, and (iv) adenylate cyclase. The surface origin of
j3-RECEPTOR~DENYLATE
cycLAsE
IN
DENERVATED
MUSCLE
363
these membranes was also confirmed by their high specific lzsI radioactivity after whole muscle fibers were subjected to lactoperoxidase iodination followed by the above subcellular fractionation procedure ( 1, 2). Protein Determination. Protein was determined by the method of Lowry et al. (14). Adenylate Cyclase Determination. Adenylate cyclase activity was measured in a final volume of 100 ~1 containing 50 InM Tris-HCl (PH 7.4), 10 mM MgCl2, 20 mM creatine phosphate, 2 mM CAMP, 20 U creatine kinase, and 1 mM ATP plus [c~-~~P]ATP (5 to 10 x lo6 cpm). Sodium fluoride, Gpp( NH)p, and catecholamines were added as described in the figure captions. The reaction was initiated by the addition of membranes (150 to 180 pg protein) preincubated at 25°C. After 5 nun, except where indicated, the reaction was terminated by the addition of 100 ~1 “stop solution,” consisting of 40 mM ATP, 2 mM CAMP, and 2% sodium dodecyl sulfate. Purification and quantitation of the [32P]~AMP produced were carried out as described by Salomon et al. (19). Reaction rates were linear with respect to time and protein concentration. ( - ) - [ 3H] Dilzydroalprenolol Binding. Binding studies were carried out in 50 mnr Tris-HCl (pH 7.4), 10 mM MgC12, and various concentrations of binding ligands. Fresh membrane material (200 PLg protein/ml) was added to duphcate sets of polycarbonate centrifuge tubes (13~ml volume) that contained (-)- [3H]‘dihydroalprenolol spanning the concentration range of 1 x lo-l1 to 1 Xl lo-? M. One set of tubes also contained isoproterenol at a final concentration of 1 X lo-” M for assessment of “nonspecific” binding. The final reaction volume for both sets of tubes was 0.5 ml. All tubes were incubated 10 to 15 min at 4°C and then duplicate 75-~1 samples were taken to determine total alprenolol concentrations. The membranes were incubated an additional 1.5 h and then centrifuged 30 min at 144,000 g, 4°C. The supernatants were transferred to glass culture tubes and duplicate 75-~1 samples were removed for determination of equilibrium or free alprenolol concentrations. Further transfers of these supernatants did not result in a significant change in alprenolol concentration and did not contain detectable protein. The concentration of bound (-) - [3H] dihydroalprenolol, VT, (total moles bound per gram membrane protein) was determined from the difference between the total and free dihydroalprenolol concentrations. “Nonspecific” binding (vNS) was determined from the linear relationship obtained in the presence of 1 x 10m3M isoproterenol and varying concentrations of ( -) - [ 3H] dihydroalprenolol. vNS values were subtracted from VT to obtain values for specific binding (I$) of dihydroalprenolol to the p-adrenergic receptor (3). We originally chose (-)-isoproterenol for this purpose because it has been shown to have greater affinity for the p-adrenergic receptor than other agonists of the adenylate
364
SMITH,
GREFRATH,
AND
APPEL
cyclase system while retaining greater structural similarity to cpinephrine relative to antagonists of greater affinity. Materials. ( - ) - [ 3H] Dihydroalprenolol (sp act 3.2 Ci/mmol) , [ (w-~~P] ATP (sp act 10 Ci/mmol), and cyclic [3H]AMP (sp act 1 to 5 Ci/mmol) were purchased from New England Nuclear Co., Boston, Massachusetts. ( -) -1soproterenol bitartrate, ( -) -epinephrine bitartrate, ( -) -norepinephrine bitartrate, creatine kinase (155 U/mg), and alumina, neutral grade, were obtained from Sigma Chemical Co., St. Louis, Missouri. Dowex SOWX2 (ZOO to 400 mesh) was from Bio-Rad. 5’-Guanylyl imidodiphosphate was purchased from ICN. All other chemicals used in this study were of reagent grade and were purchased from the usual commercial sources. RESULTS Denervation results in the modification of various biochemical properties of muscle surface membrane including decreased membrane protein phosphorylation and increased Na+K+ ( Mg2+) ATPase activity, sialoglycoprotein, and acetylcholine receptor content (1, 2, 22). After 7 to 10 days of denervation, both sodium fluoride- and isoproterenol-stimulated adenylate cyclase activities are reduced to 70% of normal. For the present studies, s-day denervation was used because the loss of enzyme activity (50% decline) by this time has not progressed to a point where experimental precision is sacrificed (70 to SO%, 7 to 10 days). A summary of selected properties of the /3-receptor-adenylate cyclase system of normal sarcolemma is included (Table 1) to facilitate comparison of data presented in this paper on denervated muscle sarcolemma (6, 7, 21) particularly in relation to Figs. 1 and 2. Kinetics of Adenylate Cyclase Activation. The time course of activation of adenylate cyclase in denervated sarcolemma is qualitatively similar to that observed in normal sarcolemma. The rate of reaction deviated from linearity after 5 min for basal, Gpp(NH)p-, isoproterenol-, and isoproterenol plus Gpp( NH)p-activated states of the enzyme (Fig. 1). Sodium fluoride (2 x 1O-2 M) activation remained linear for the 15-min duration of the experiment. The reason for this difference in linearity between NaF and the other effecters is not known. It is not due to limitation of ATP because the addition of ATP at the IO-min time point did not cause an increase in either basal or isoproterenol activity. Further, it was shown in this laboratory that both basal and isoproterenol activation are not altered after 1 h incubation at 25”C, indicating that in the present study selective temperature inactivation was not responsible for the difference in linearity in the presence of NaF or isoproterenol. The degree of activation of adenylate cyclase in denervated sarcolemma also was qualitatively similar to that observed in normal sarcolemma (Fig.
f3-RECEPTOR-ADENYLATE
CYCLASE
TABLE Properties
of the @-Receptor-Adenylate Normal Skeletal Muscle Property
+GPPWWP
&Receptor K N
DE~~ERVATED
~~IIUSCLE
365
1 Cyclase Sarcolemmaa
System
of
Value
Adenylate cyclase activity Kinetics Basal Gpp(NH)p (1 X 1O-4 M) Isoproterenol (1 X 10e7 M) NaF (2 X lo-* Biological potency Isoproterenol Epinephrine Norepinephrine
IN
M)
51 250 (5) 160 (3) 551 (10) 1750 (34)
(C,) 1.9 x 1o-7 1.2 x 1o-6 1.8 X 1fF
M M M
3.3 X lo-l0 mol/liter 1.1 X 10m9 mol/g Membrane protein
Q This table is a summary of results taken from a manuscript submitted for publication by Grefrath, S. H., Smith, P. B., and Appel, S. H. For kinetic data, reactions were carried out for 15 min at the indicated concentrations of isoproterenol and Gpp(NH)p. Values are expressed as picomoles CAMP formed/S min/mg membrane protein. Numbers in parentheses indicate the fold stimulation above basal enzyme activity. Biological potency of the catecholamines was determined for 1 X lo”‘- to 1 X 10M4 M hormone. C+ signifies the concentration of hormone required to half-maximally stimulate enzyme activity. B-Receptor studies were carried out by examining the binding of (-)-pH]dihydroalprenolol at concentrations of 1 X 10-l’ to 1 X lo-’ M. g (affinity constant) and N (number of binding sites) were determined by Scatchard analysis of the equilibrium binding data after correction for nonspecific binding.
1). Sodium fluoride gave the greatest activation (900 pmol CAMP/S min/ mg) corresponding to a 45fold increase from basal activity. Isoproterenol and Gpp(NH)p activated adenylate cyclase 4.Z and 3.5-fold. In combination, Gpp (NH)p potentiated isoproterenol-stimulated activity about twofold, indicating the operation of guanylyl nucleotide synergism in the activation of adenylate cyclase by isoproterenol. The major difference between normal and denervated sarcolemmal enzyme was the specific activity in the presence of effecters and basal activity. Comparing values given in Table 1 to those of the 5-min time points in Fig. 1 shows an approximate 50% decline of basal, NaF-, or isoproterenol-stimulated activity and a 60% decrease in Gpp (NH) p-stimulated activity. Potency Series. Skeletal muscle classically displays a ,&-type adrenergic response to the catecholamine hormones (16). Isoproterenol is more effec-
366
SMITH,
GREFRATH,
5 TIME
AND
IO (mlnl
APPEL
15
FIG. 1. Kinetics of adenylate cyclase activation. Adenylate cyclase activity was measured as described in Methods and Materials at the indicated times. Basal (0-O ; Gpp(NH)p, 1 X 10e4 M (O-O) ; isoproterenol, 1 X IO-’ M (&---A) ; sodium fluoride, 2 X lo-’ M ( m---m) ; isoproterenol, 1 X IO-’ M, plus Gpp( NH)p, 1 X lo-’ M (&--A). Determinations were performed in duplicate for at least three separate membrane preparations.
tive than epinephrine and norepinephrine in stimulating CAMP accumulation and glycogenolysis. Recent reports showing the interconversion of catecholamine potency in normal vs. transformed hepatocytes indicate that the physiologic state of the cell may influence the specificity of hormonal action (10). Denervated muscle sarcolemma retained the &-type specificity
2. Catecholamine potency series. Adenylate cyclase activity was measured as in Methods and Materials at the indicated hormone concentrations. Values the hormone-stimulated activity minus basal activity : isoproterenol (A-A), epinephrine (A-A), norepinephrine (0-O). Determinations were in duplicate for at least three separate membrane preparations. FIG.
described represent
P-RECEPTOR-ADENYLATE
CYCLASE
-10
-9
-8 -7 log Clsoproierenoll
IN
DENERVATED
-6
MUSCLE
367
-5
FIG. 3. Comparison of Gpp(NH)p effect on isoproterenol activation of adenylate cyclase of normal and denervated muscle sarcolemma. Adenylate cyclase activity was measured as described in Methods and Materials. Normal, isoproterenol plus Gpp(NH)p, 1 X 10m4 M (0-O) ; normal, isoproterenol alone (O--O) ; denervated, isoproterenol plus Gpp(NH)p, 1 X lo-” M (A-A) ; denervated, isoproterenol alone (n----n). Determinations were in duplicate for at least three separate membrane preparations.
for adenylate cyclase activation. The concentrations at which the catecholamines gave half-maximal stimulation (C,) were 9.3 X lo-*, 2.5 X 10e7, and 2.8 X lO-‘j M, respectively, for isoproterenol, epinephrine, and norepinephrine. The discrimination between isoproterenol and epinephrine was not as great as the lo-fold difference observed in normal sarcolemma, and the C+ was lower for each catecholamine compared to normal sarcolemmal enzyme activity (Table 1) . The specific activity of adenylate cyclase in the presence of each catecholamine was decreased to 50% of normal in denervated sarcolemma. The maximum activation of the enzyme occurred at concentrations of 5 X 10e8, 1 x 10m5, and 1 x 10m4 M, respectively, for isoproterenol, epinephrine, and norepinephrine in denervated sarcolemma. In normal sarcolemma, these maximal values were, respectively, 5 x 10-6, 1 x 10-4, and 5 x 10m4M. The differences in adenylate cyclase activation between normal and denervated sarcolemma at the maximum activating concentrations of the catecholamines were (normal vs. denervated) : isoproternol, 320 vs. 175 ; epinephrine, 320 vs. 180; and norepinephrine, 210 vs. 130 (values expressed as pmol CAMP/~ min/mg). This finding indicates that denervation results in a uniform decline in catecholamine hormone activation. The data also suggest that the response to epinephrine, the physiological hormone, although decreased is more sensitive in denervated sarcolemma because the concentration of epinephrine required for half-maximal and maximal stimulation of adenylate cyclase is almost lo-fold lower than that of normal enzyme.
368
SMITH,
GREFRATH,
AND
APPEL
cyclase systems Efiect of Gpp(NH)p. A number of /?-receptor-adenylate have been shown to be modulated by the guanylyl nucleotides. The basic finding in the erythrocyte membrane is that Gpp (NH) p causes a stimulation of catecholamine activation of adenylate cyclase and decreases the Cl for activation by approximately lo-fold (11, 13). This property also exists for the stimulation of adenylate cyclase activity by isoproterenol in the presence and absence of optimum concentrations (1 x 10m4 M) of Gpp(NH) p, 5’-Guanylyl imi’dodiphosphate shifted the C* from 1.5 X 1O-T to 2 x 1O-8 M for normal and from 1 X 1O-7 to 4 X 1O-s M for denervated sarcolemmal enzyme activation. In addition, the maximum activation was increased 2- and 1.5-fold for normal and denervated adenylate cyclase. The main difference noted between normal and denervated sarcolemmal enzyme was the decreased specific activity in denervated sarcolemma whether Gpp( NH)p was present or not. ,&Adrenergic Receptor. The 50% decline in basal and NaF-activated adenylate cyclase in denervated sarcolemma suggested that a loss of enzyme had occurred. To verify this possibility, p-receptor content and affinity were assessed (Fig. 4). Binding studies using the p-adrenergic antagonist (-)- [3H] dihydroalprenolol demonstrated that normal and denervated sarcolemma do not differ with respect to concentration-dependent equilibrium binding of this ligand. Iterative curve fitting of data from four preparations of normal sarcolemma yielded a dissociation constant of 3.3 f 0.4 x lo-lo mol/liter and a value of N (number of sites) of 1.1 t 0.06 x 1O-D mol/g membrane protein. The corresponding values for denervated sarcolemma were 2.8 f 0.2 XI lo-lo mol/liter and 1.23 rfI 0.07 X 10-D mol/g membrane protein. Dihydroalprenolol binding in both membrane
1.2 2 u
1.0
z c
0.8
,$
0.6 0.4
-10
-9
log [Dihydroolprenolol]
FIG. 4. Binding of [‘H]dihydroalprenolol to normal and denervated muscle sarcolemma. Binding studies were done at equilibrium at the indicated Iigand concentrations as described in Methods and Materials. Normal (O-O), denervated (n-0). Determinations were in duplicate for at least four separate membrane preparations.
P-RECEPTOR~DENYLATE
c~cL.4s~
IN
DENERVATED
hfusc~~
369
preparations had the same potency series and stereospecificity for catecholamines as the adenylate cyclase enzyme. These data suggest that the physiologic p-receptor is not altered in the denervated state, thus indicating that loss of hormone-stimulated adenylate cyclase activity in denervated sarcolemma is not due to loss of ability to bind hormone. DISCUSSION Denervation of skeletal muscle is characterized by several metabolic alterations which are influenced by metabolic hormones. These alterations include (i) decreased protein synthesis, glycogen stores, rate of glycogen utilization, and ATP content, and (ii) increased lysosomal enzyme activity and rate of protein degradation (5, 8). The basis for the present work was the finding that catecholamine hormone-activated adenylate cyclase activity diminished progressively after denervation. Commencing between Days 3 and 4, activity diminished to 50% of normal muscle sarcolemma by Day 5 of denervation. In this paper we analyzed three aspects of the membrane component of the /?-adrenergic response after muscle denervation. It was shown that the thermodynamic properties of the p-receptor, modulation of adenylate cyclase activity by Gpp (NH) p, and catecholamine hormone specificity were essentially unchanged by denervation. The basic alteration, i.e., 50% decrease in basal, NaF-, Gpp (NH)p-, and catecholamine-stimulated adenylate cyclase, appears to be due to loss of active enzyme. The fact that the loss of Gpp (NH) p-stimulated and basal activity was equivalent provides additional evidence that Gpp( NH)p action is exerted at the enzyme and not the receptor level (17, 20). These findings are similar to those made on developing systems. Amphibian erythrocytes studied from the tadpole stage possess basal and NaF-stimulated adenylate cyclase activity, but little catecholamine receptor and catecholamine-stimulated adenylate cyclase. As metamorphosis progresses, receptor content increases and catecholamine activation of adenylate cyclase becomes prominent (12, 18). Denervation of mammalian skeletal muscle appears to induce a process opposite to that seen for development since adenylate cyclase activity is lost without change of p-receptor content and affinity for catecholamines. These data suggest that receptor and adenylate cyclase behave as separate units not under coordinate control in terms of formation of a functional membrane component of the ,&adrenergic response. The physiological significance of these findings needs clarification. It was reported that CAMP concentrations in denervated muscle do not decline as does adenylate cyclase activity (4). In our laboratory we measured the isoproterenol-induced increase in CAMP in normal and denervated (Sday) extensor digitorum longus muscle. The increase in CAMP is depressed by
370
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GREFRATH,
AND
APPEL
40% in denervated muscle, suggesting that the membrane studies presented here accurately reflect the physiologic response in the intact tissue (J. Blosser, unpublished results). It also is important to consider other parts of the response when attempting to correlate membrane studies to the physiologic response. Thus, alterations in phosphodiesterase activity, intracellular calcium and guanylyl nucleotide concentrations, and specific protein activators of adenylate cyclase could play a role in any alteration of a CAMP-mediated process (15). These factors must be considered in attempting to evaluate the physiologic consequences of denervation on the ,&adrenergic response. One finding of potential value of this study relates to the inhibitory action of the catecholamines on skeletal muscle protein catabolism (9). The loss of the p-adrenergic response with denervation that we observed may represent an initial event in the increased protein catabolism with the attendant loss of muscle weight characteristic of the denervated state of skeletal muscle. REFERENCES 1. 2.
3.
4. 5.
6.
7.
8.
9.
10.
11.
C. G., AND S. H. APPEL. 1973. Macromolecular characterization of muscle membranes. J. Biol. Chewz. 248 : 5156-5163. ANDREW, C. G., R. R. ALMON, AND S. H. APPEL. 1974. Macromolecular characterization of muscle membranes: Acetylcholine receptor of normal and denervated muscle. J. Biol. Chew 249: 6163-6165. ATLAS, D., M. L. STEER, AND A. LEVITZKI. 1974. Stereospecific binding of propranolol and catecholamines to the p-adrenergic receptor. Proc. Natl. Acad. Sci. U.S.A. 71: 4246-4248. CARLSEN, R. C. 1975. The possible role of cyclic AMP in the neurotrophic control of skeletal muscle. J. Physiol. (London) 247: 343-361. GOLDBERG, A. L., C. JABLECKI, AND J. B. LI. 1947. Effects of use and disuse on amino acid transport and protein turnover in muscle. Ann. N.Y. Acad. Sci. 228: 190-201. GREFRATH, S. P., P. B. SMITH, AND S. H. APPEL. 1976. p-Adrenergic receptor and catecholamine stimulated adenyl cyclase in normal and denervated skeletal muscle sarcolemma. Neurosci. Abstr. 2: 466. GREFRATH, S. P., P. B. SMITH, AND S. H. APPEL. 1977. @-Adrenergic receptor and adenyl cyclase in mammalian muscle sarcolemma. Arch. Biochem. Biophys. (Submitted). GUTMANN, E. 1976. Neurotrophic relations. Anuu. Rev. Physiol. 38 : 177-216. KARL, I. E., A. J. GARBER, AND D. M. KIPNIS. 1976. Alanine and glutamine synthesis and release from skeletal muscle. III. Dietary and hormonal regulation. J. Biol. Chew 251: 844-850. LACOMBE, M. L., E. RENE, G. GUELLAN, AND J. HANOUNE. 1976. Transformation of the & adrenoceptor in normal rat liver into p1 type in Zajdela hepatoma. Nature (London) 262 : 70-72. LEFKOWITZ, R. J., AND M. G. CARON. 1975. Characteristics of S-guanylyl imidodiphosphate activated adenylate cyclase. J. Biol. Chem. 251) : 4418-4422. ANDREW,
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CycLAsE
IN DENERVATED
MUSCLE
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12. LEFKOWITZ, R. J,, L. E. LIMBIRD, C. MUKHERJEE, AND M. G. CARON. 1976.The P-adrenergicreceptorand adenylatecyclase.Biochim.Biopkys. Acta 457: 1-39. 13. LEVITZKI, A., N. SEVILLA, D. ATLAS, AND M. L. STEER. 197.5. Ligand specificity and characteristics of the /3-adrenergic receptor in turkey erythrocyte plasma membranes. J. Mol. Biol. 97: 35-53. 14. LOWRY, 0. H., N. J. ROSEBROLJGH, A. L. FARR, AND R. J. RANDALL. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chcrrt. 193: 265-275. 15. LYNCH, T. J., E. A. TALLANT, AND U. Y. CHEUNY. 1976. Stimulation of rat brain adenylate cyclase by an endogenous protein activator. Fed. Proc. 35: 1633. 16. NARAHARA, H. T., AND C. F. CORI. 1968. Hormonal control of carbohydrate metabolism in muscle. Pages 375-391 in F. DICKENS, P. J. RANDLE, AND W. J. WHELAN, Ed., Carbohydrate Metabolism a?ld Its Disorders Academic Press, New York. 17. RODBELL, M., M. C. LIN, Y. SALOMON, C. LONDOS, J. P. HARWOOD, B. R. MARTIN, M. RENDELL, AND M. BERMAN. 1975. Role of adenine and guanine nucleotides in the activity and response of adenylate cyclase systems to hormones : Evidence for multisite transition states. Pages 3-30 in G. I. DRUMMOND, P. GREENGARD, AND G. A. ROBISON, Ed., Advances in Cyclic Nucleotide Research, Vol. 5. Raven Press, New York. 18. ROSEN, 0. M., AND S. N. ROSEN. 1968. The effect of catecholamines on the adenyl cyclase of frog and tadpole hemolysates. Biochem. Biophys. lies. Commun. 31: 82-91. 19. SALOMON, V., C. LONDOS, AND M. RODBELL. 1974. A highly sensitive adenylate cyclase assay. A?tal. Biochem. 58 : 541-548. 20. SEVILLA, N., M. L. STEER, AND LEVITZKI. 1976. Synergistic activation of adenylate cyclase by guanylyl imidophosphate and epinephrine. Biochemistry 15 : 349334$8. 21. SILZITH, P. B., AND S. H. APPEL. 1976. Biochemical analysis of surface membrane alterations in denervated mammalian muscle. Fed. Proc. 35: 1652. 22. SMITH, P. B., AND S. H. APPEL. 1977. Development of denervation alterations in surface membranes of mammalian skeletal muscle. Exfi. Neurol. 56: 102-114. 23. SMITH, P. B., AND S. H. APPEL. 1977. Isolation and characterization of the surface membranes of fast and slow mammalian skeletal muscle. Biochim. Biophys. Acta 466 : 109-122.
NEUROLOGY
59,
361-371
(1978)
P-Adrenergic Receptor-Adenylate Denervated Sarcolemmal P.
BLAISE
SMITH,
STUART
P.
GREFRATH,
Cyclase Membrane AND
STANLEY
of
H.
APPEL
l
Departme& of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27103, and Division of Neurology, Baylor College of Medicine, Houston. Texas 77025 Received
September
27,1977;
received December
revision
2,1977
Five days after motor denervation, mammalian skeletal muscle sarcolemma undergoes a 50% decline in basal and catecholamine-stimulated adenylate cyclase activity. Sodium fluoride- and S-guanylyl imidophosphate [ Gpp( NH) p) ]-stimulated activities also are depressed. However, properties of the /%adrenergic receptor in the plasma membrane preparation of denervated muscle are similar to those of normal innervated muscle. The number and affinity of specific (-) -[3H]dihydroalprenolol binding sites, the effects of catecholaminergic ligands on binding (isoproterenol > epinephrine > norepinephrine), the stimulatory effects of Gpp(NH)p on adenylate cyclase activity, and the shift in concentration of catecholamines required to activate adenylate cyclase in the presence of Gpp(NH)p were similar in normal and denervated membranes. Thus denervation appears to uncouple the adenylate cyclase response from p-adrenergic stimulation primarily by a loss of adenylate cyclase activity with no change in receptor properties.
INTRODUCTION Previous work from our laboratory characterized the ,8-adrenergic receptor-adenylate cyclase system in the sarcolemmal membrane isolated l This paper is dedicated to the memory of Dr. Stuart P. Grefrath, deceased. We thank Mrs. Tommye Campbell for expert secretarial assistance. This work was supported by Grant NS-07872 from the National Institutes of Health. Portions of this study were presented by Dr. P. Blaise Smith at the annual FASEB meeting, San Francisco, California, June 1976, and Dr. Stuart P. Grefrath at the annual meeting of the Society for Neuroscience, Toronto, Ontario, Canada, November 1976. Requests for reprints should be addressed to Dr. Appel at Baylor College of Medicine. Abbreviations : Gpp (NH) p--5’-guanylyl imidodiphosphate ; CAMP-adenosine 3’ : 5’-cyclic monophosphate. 361 0014-4886/78/0593-0361$02.00/O All
Copyright 0 1978 by Academic Press, Inc. rights of reproduction in any form reserved.
362
SMITH,
GREFRATH,
AND
APPEL
from normal mammalian skeletal muscle (6, 7, 21). In these membranes adenylate cyclase activity was stimulated proportionately to the affinity of catecholamine hormone binding to the p-receptor. The kinetic properties of the enzyme and dihydroalprenoiol binding properties of the receptor suggested the presence of a coupled Ps-type adrenergic response similar to the physiological and pharmacological response of skeletal muscle to catecholamines. In the course of studies on the effect of denervation on the biochemical properties of the sarcolemma, it was found that catecholamine hormone stimulation of adenylate cyclase activity declined 50% after 5 days of denervation (22). This observation plus our characterization of the P-receptor, adenylate cyclase, and guanylyl nucleotide effect in normal muscle prompted the present investigation in which we examine each of these sarcolemmal components of the p-adrenergic response in denervated muscle. The object of this study was to determine whether loss of receptor or impairment of active enzyme was responsible for the diminution of catecholamine hormone-stimulated adenylate cyclase activity. The data suggest that denervation results in the loss of active enzyme and does not alter either receptor properties or guanylyl nucleotide regulation. METHODS
AND
MATERIALS
Denervation. Denervation was by excision of a 2-cm segment of the sciatic nerve from the midthigh region of female (200 to 250-g) Wistar rats. Sarcolemmal Membrane Isolation. An enriched fraction of the sarcolemma1 membrane was prepared from 5-day denervated muscle as previously described (1, 23). Briefly, rat lower limb muscle was minced and homogenized (Polytron, Brinkman Instrument Co.) in buffer consisting of 0.25 M sucrose, 0.2 mM Naa EDTA, and 0.2 mM Tris-HCl, pH 7.5. The homogenate was centrifuged 10 min at 1000 g and the pellet (nuclear fraction) retained for further hypertonic salt extractions. Crude membrane material resulting from this extraction procedure was subjected to continuous (15 to 35%) sucrose gradients and centrifugation was carried out at 200,000 g for 210 min. Membrane material distributing in the 19 to 24% zone of the gradient was removed, concentrated by centrifugation (48,000 g, 120 min) , and resuspended in 1 mM Tris-HCl buffer, pH 7.5, to a protein concentration of 2 to 4 mg/ml. All operations were carried out at 4°C. p-Receptor and adenylate cyclase studies were done immediately. This membrane preparation is characterized by possessing the highest specific and total activities of (i) Na+K+ (Mg*+) ATPase, (ii) sialic acid content, (iii) acetylcholine receptor, and (iv) adenylate cyclase. The surface origin of
j3-RECEPTOR~DENYLATE
cycLAsE
IN
DENERVATED
MUSCLE
363
these membranes was also confirmed by their high specific lzsI radioactivity after whole muscle fibers were subjected to lactoperoxidase iodination followed by the above subcellular fractionation procedure ( 1, 2). Protein Determination. Protein was determined by the method of Lowry et al. (14). Adenylate Cyclase Determination. Adenylate cyclase activity was measured in a final volume of 100 ~1 containing 50 InM Tris-HCl (PH 7.4), 10 mM MgCl2, 20 mM creatine phosphate, 2 mM CAMP, 20 U creatine kinase, and 1 mM ATP plus [c~-~~P]ATP (5 to 10 x lo6 cpm). Sodium fluoride, Gpp( NH)p, and catecholamines were added as described in the figure captions. The reaction was initiated by the addition of membranes (150 to 180 pg protein) preincubated at 25°C. After 5 nun, except where indicated, the reaction was terminated by the addition of 100 ~1 “stop solution,” consisting of 40 mM ATP, 2 mM CAMP, and 2% sodium dodecyl sulfate. Purification and quantitation of the [32P]~AMP produced were carried out as described by Salomon et al. (19). Reaction rates were linear with respect to time and protein concentration. ( - ) - [ 3H] Dilzydroalprenolol Binding. Binding studies were carried out in 50 mnr Tris-HCl (pH 7.4), 10 mM MgC12, and various concentrations of binding ligands. Fresh membrane material (200 PLg protein/ml) was added to duphcate sets of polycarbonate centrifuge tubes (13~ml volume) that contained (-)- [3H]‘dihydroalprenolol spanning the concentration range of 1 x lo-l1 to 1 Xl lo-? M. One set of tubes also contained isoproterenol at a final concentration of 1 X lo-” M for assessment of “nonspecific” binding. The final reaction volume for both sets of tubes was 0.5 ml. All tubes were incubated 10 to 15 min at 4°C and then duplicate 75-~1 samples were taken to determine total alprenolol concentrations. The membranes were incubated an additional 1.5 h and then centrifuged 30 min at 144,000 g, 4°C. The supernatants were transferred to glass culture tubes and duplicate 75-~1 samples were removed for determination of equilibrium or free alprenolol concentrations. Further transfers of these supernatants did not result in a significant change in alprenolol concentration and did not contain detectable protein. The concentration of bound (-) - [3H] dihydroalprenolol, VT, (total moles bound per gram membrane protein) was determined from the difference between the total and free dihydroalprenolol concentrations. “Nonspecific” binding (vNS) was determined from the linear relationship obtained in the presence of 1 x 10m3M isoproterenol and varying concentrations of ( -) - [ 3H] dihydroalprenolol. vNS values were subtracted from VT to obtain values for specific binding (I$) of dihydroalprenolol to the p-adrenergic receptor (3). We originally chose (-)-isoproterenol for this purpose because it has been shown to have greater affinity for the p-adrenergic receptor than other agonists of the adenylate
364
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APPEL
cyclase system while retaining greater structural similarity to cpinephrine relative to antagonists of greater affinity. Materials. ( - ) - [ 3H] Dihydroalprenolol (sp act 3.2 Ci/mmol) , [ (w-~~P] ATP (sp act 10 Ci/mmol), and cyclic [3H]AMP (sp act 1 to 5 Ci/mmol) were purchased from New England Nuclear Co., Boston, Massachusetts. ( -) -1soproterenol bitartrate, ( -) -epinephrine bitartrate, ( -) -norepinephrine bitartrate, creatine kinase (155 U/mg), and alumina, neutral grade, were obtained from Sigma Chemical Co., St. Louis, Missouri. Dowex SOWX2 (ZOO to 400 mesh) was from Bio-Rad. 5’-Guanylyl imidodiphosphate was purchased from ICN. All other chemicals used in this study were of reagent grade and were purchased from the usual commercial sources. RESULTS Denervation results in the modification of various biochemical properties of muscle surface membrane including decreased membrane protein phosphorylation and increased Na+K+ ( Mg2+) ATPase activity, sialoglycoprotein, and acetylcholine receptor content (1, 2, 22). After 7 to 10 days of denervation, both sodium fluoride- and isoproterenol-stimulated adenylate cyclase activities are reduced to 70% of normal. For the present studies, s-day denervation was used because the loss of enzyme activity (50% decline) by this time has not progressed to a point where experimental precision is sacrificed (70 to SO%, 7 to 10 days). A summary of selected properties of the /3-receptor-adenylate cyclase system of normal sarcolemma is included (Table 1) to facilitate comparison of data presented in this paper on denervated muscle sarcolemma (6, 7, 21) particularly in relation to Figs. 1 and 2. Kinetics of Adenylate Cyclase Activation. The time course of activation of adenylate cyclase in denervated sarcolemma is qualitatively similar to that observed in normal sarcolemma. The rate of reaction deviated from linearity after 5 min for basal, Gpp(NH)p-, isoproterenol-, and isoproterenol plus Gpp( NH)p-activated states of the enzyme (Fig. 1). Sodium fluoride (2 x 1O-2 M) activation remained linear for the 15-min duration of the experiment. The reason for this difference in linearity between NaF and the other effecters is not known. It is not due to limitation of ATP because the addition of ATP at the IO-min time point did not cause an increase in either basal or isoproterenol activity. Further, it was shown in this laboratory that both basal and isoproterenol activation are not altered after 1 h incubation at 25”C, indicating that in the present study selective temperature inactivation was not responsible for the difference in linearity in the presence of NaF or isoproterenol. The degree of activation of adenylate cyclase in denervated sarcolemma also was qualitatively similar to that observed in normal sarcolemma (Fig.
f3-RECEPTOR-ADENYLATE
CYCLASE
TABLE Properties
of the @-Receptor-Adenylate Normal Skeletal Muscle Property
+GPPWWP
&Receptor K N
DE~~ERVATED
~~IIUSCLE
365
1 Cyclase Sarcolemmaa
System
of
Value
Adenylate cyclase activity Kinetics Basal Gpp(NH)p (1 X 1O-4 M) Isoproterenol (1 X 10e7 M) NaF (2 X lo-* Biological potency Isoproterenol Epinephrine Norepinephrine
IN
M)
51 250 (5) 160 (3) 551 (10) 1750 (34)
(C,) 1.9 x 1o-7 1.2 x 1o-6 1.8 X 1fF
M M M
3.3 X lo-l0 mol/liter 1.1 X 10m9 mol/g Membrane protein
Q This table is a summary of results taken from a manuscript submitted for publication by Grefrath, S. H., Smith, P. B., and Appel, S. H. For kinetic data, reactions were carried out for 15 min at the indicated concentrations of isoproterenol and Gpp(NH)p. Values are expressed as picomoles CAMP formed/S min/mg membrane protein. Numbers in parentheses indicate the fold stimulation above basal enzyme activity. Biological potency of the catecholamines was determined for 1 X lo”‘- to 1 X 10M4 M hormone. C+ signifies the concentration of hormone required to half-maximally stimulate enzyme activity. B-Receptor studies were carried out by examining the binding of (-)-pH]dihydroalprenolol at concentrations of 1 X 10-l’ to 1 X lo-’ M. g (affinity constant) and N (number of binding sites) were determined by Scatchard analysis of the equilibrium binding data after correction for nonspecific binding.
1). Sodium fluoride gave the greatest activation (900 pmol CAMP/S min/ mg) corresponding to a 45fold increase from basal activity. Isoproterenol and Gpp(NH)p activated adenylate cyclase 4.Z and 3.5-fold. In combination, Gpp (NH)p potentiated isoproterenol-stimulated activity about twofold, indicating the operation of guanylyl nucleotide synergism in the activation of adenylate cyclase by isoproterenol. The major difference between normal and denervated sarcolemmal enzyme was the specific activity in the presence of effecters and basal activity. Comparing values given in Table 1 to those of the 5-min time points in Fig. 1 shows an approximate 50% decline of basal, NaF-, or isoproterenol-stimulated activity and a 60% decrease in Gpp (NH) p-stimulated activity. Potency Series. Skeletal muscle classically displays a ,&-type adrenergic response to the catecholamine hormones (16). Isoproterenol is more effec-
366
SMITH,
GREFRATH,
5 TIME
AND
IO (mlnl
APPEL
15
FIG. 1. Kinetics of adenylate cyclase activation. Adenylate cyclase activity was measured as described in Methods and Materials at the indicated times. Basal (0-O ; Gpp(NH)p, 1 X 10e4 M (O-O) ; isoproterenol, 1 X IO-’ M (&---A) ; sodium fluoride, 2 X lo-’ M ( m---m) ; isoproterenol, 1 X IO-’ M, plus Gpp( NH)p, 1 X lo-’ M (&--A). Determinations were performed in duplicate for at least three separate membrane preparations.
tive than epinephrine and norepinephrine in stimulating CAMP accumulation and glycogenolysis. Recent reports showing the interconversion of catecholamine potency in normal vs. transformed hepatocytes indicate that the physiologic state of the cell may influence the specificity of hormonal action (10). Denervated muscle sarcolemma retained the &-type specificity
2. Catecholamine potency series. Adenylate cyclase activity was measured as in Methods and Materials at the indicated hormone concentrations. Values the hormone-stimulated activity minus basal activity : isoproterenol (A-A), epinephrine (A-A), norepinephrine (0-O). Determinations were in duplicate for at least three separate membrane preparations. FIG.
described represent
P-RECEPTOR-ADENYLATE
CYCLASE
-10
-9
-8 -7 log Clsoproierenoll
IN
DENERVATED
-6
MUSCLE
367
-5
FIG. 3. Comparison of Gpp(NH)p effect on isoproterenol activation of adenylate cyclase of normal and denervated muscle sarcolemma. Adenylate cyclase activity was measured as described in Methods and Materials. Normal, isoproterenol plus Gpp(NH)p, 1 X 10m4 M (0-O) ; normal, isoproterenol alone (O--O) ; denervated, isoproterenol plus Gpp(NH)p, 1 X lo-” M (A-A) ; denervated, isoproterenol alone (n----n). Determinations were in duplicate for at least three separate membrane preparations.
for adenylate cyclase activation. The concentrations at which the catecholamines gave half-maximal stimulation (C,) were 9.3 X lo-*, 2.5 X 10e7, and 2.8 X lO-‘j M, respectively, for isoproterenol, epinephrine, and norepinephrine. The discrimination between isoproterenol and epinephrine was not as great as the lo-fold difference observed in normal sarcolemma, and the C+ was lower for each catecholamine compared to normal sarcolemmal enzyme activity (Table 1) . The specific activity of adenylate cyclase in the presence of each catecholamine was decreased to 50% of normal in denervated sarcolemma. The maximum activation of the enzyme occurred at concentrations of 5 X 10e8, 1 x 10m5, and 1 x 10m4 M, respectively, for isoproterenol, epinephrine, and norepinephrine in denervated sarcolemma. In normal sarcolemma, these maximal values were, respectively, 5 x 10-6, 1 x 10-4, and 5 x 10m4M. The differences in adenylate cyclase activation between normal and denervated sarcolemma at the maximum activating concentrations of the catecholamines were (normal vs. denervated) : isoproternol, 320 vs. 175 ; epinephrine, 320 vs. 180; and norepinephrine, 210 vs. 130 (values expressed as pmol CAMP/~ min/mg). This finding indicates that denervation results in a uniform decline in catecholamine hormone activation. The data also suggest that the response to epinephrine, the physiological hormone, although decreased is more sensitive in denervated sarcolemma because the concentration of epinephrine required for half-maximal and maximal stimulation of adenylate cyclase is almost lo-fold lower than that of normal enzyme.
368
SMITH,
GREFRATH,
AND
APPEL
cyclase systems Efiect of Gpp(NH)p. A number of /?-receptor-adenylate have been shown to be modulated by the guanylyl nucleotides. The basic finding in the erythrocyte membrane is that Gpp (NH) p causes a stimulation of catecholamine activation of adenylate cyclase and decreases the Cl for activation by approximately lo-fold (11, 13). This property also exists for the stimulation of adenylate cyclase activity by isoproterenol in the presence and absence of optimum concentrations (1 x 10m4 M) of Gpp(NH) p, 5’-Guanylyl imi’dodiphosphate shifted the C* from 1.5 X 1O-T to 2 x 1O-8 M for normal and from 1 X 1O-7 to 4 X 1O-s M for denervated sarcolemmal enzyme activation. In addition, the maximum activation was increased 2- and 1.5-fold for normal and denervated adenylate cyclase. The main difference noted between normal and denervated sarcolemmal enzyme was the decreased specific activity in denervated sarcolemma whether Gpp( NH)p was present or not. ,&Adrenergic Receptor. The 50% decline in basal and NaF-activated adenylate cyclase in denervated sarcolemma suggested that a loss of enzyme had occurred. To verify this possibility, p-receptor content and affinity were assessed (Fig. 4). Binding studies using the p-adrenergic antagonist (-)- [3H] dihydroalprenolol demonstrated that normal and denervated sarcolemma do not differ with respect to concentration-dependent equilibrium binding of this ligand. Iterative curve fitting of data from four preparations of normal sarcolemma yielded a dissociation constant of 3.3 f 0.4 x lo-lo mol/liter and a value of N (number of sites) of 1.1 t 0.06 x 1O-D mol/g membrane protein. The corresponding values for denervated sarcolemma were 2.8 f 0.2 XI lo-lo mol/liter and 1.23 rfI 0.07 X 10-D mol/g membrane protein. Dihydroalprenolol binding in both membrane
1.2 2 u
1.0
z c
0.8
,$
0.6 0.4
-10
-9
log [Dihydroolprenolol]
FIG. 4. Binding of [‘H]dihydroalprenolol to normal and denervated muscle sarcolemma. Binding studies were done at equilibrium at the indicated Iigand concentrations as described in Methods and Materials. Normal (O-O), denervated (n-0). Determinations were in duplicate for at least four separate membrane preparations.
P-RECEPTOR~DENYLATE
c~cL.4s~
IN
DENERVATED
hfusc~~
369
preparations had the same potency series and stereospecificity for catecholamines as the adenylate cyclase enzyme. These data suggest that the physiologic p-receptor is not altered in the denervated state, thus indicating that loss of hormone-stimulated adenylate cyclase activity in denervated sarcolemma is not due to loss of ability to bind hormone. DISCUSSION Denervation of skeletal muscle is characterized by several metabolic alterations which are influenced by metabolic hormones. These alterations include (i) decreased protein synthesis, glycogen stores, rate of glycogen utilization, and ATP content, and (ii) increased lysosomal enzyme activity and rate of protein degradation (5, 8). The basis for the present work was the finding that catecholamine hormone-activated adenylate cyclase activity diminished progressively after denervation. Commencing between Days 3 and 4, activity diminished to 50% of normal muscle sarcolemma by Day 5 of denervation. In this paper we analyzed three aspects of the membrane component of the /?-adrenergic response after muscle denervation. It was shown that the thermodynamic properties of the p-receptor, modulation of adenylate cyclase activity by Gpp (NH) p, and catecholamine hormone specificity were essentially unchanged by denervation. The basic alteration, i.e., 50% decrease in basal, NaF-, Gpp (NH)p-, and catecholamine-stimulated adenylate cyclase, appears to be due to loss of active enzyme. The fact that the loss of Gpp (NH) p-stimulated and basal activity was equivalent provides additional evidence that Gpp( NH)p action is exerted at the enzyme and not the receptor level (17, 20). These findings are similar to those made on developing systems. Amphibian erythrocytes studied from the tadpole stage possess basal and NaF-stimulated adenylate cyclase activity, but little catecholamine receptor and catecholamine-stimulated adenylate cyclase. As metamorphosis progresses, receptor content increases and catecholamine activation of adenylate cyclase becomes prominent (12, 18). Denervation of mammalian skeletal muscle appears to induce a process opposite to that seen for development since adenylate cyclase activity is lost without change of p-receptor content and affinity for catecholamines. These data suggest that receptor and adenylate cyclase behave as separate units not under coordinate control in terms of formation of a functional membrane component of the ,&adrenergic response. The physiological significance of these findings needs clarification. It was reported that CAMP concentrations in denervated muscle do not decline as does adenylate cyclase activity (4). In our laboratory we measured the isoproterenol-induced increase in CAMP in normal and denervated (Sday) extensor digitorum longus muscle. The increase in CAMP is depressed by
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AND
APPEL
40% in denervated muscle, suggesting that the membrane studies presented here accurately reflect the physiologic response in the intact tissue (J. Blosser, unpublished results). It also is important to consider other parts of the response when attempting to correlate membrane studies to the physiologic response. Thus, alterations in phosphodiesterase activity, intracellular calcium and guanylyl nucleotide concentrations, and specific protein activators of adenylate cyclase could play a role in any alteration of a CAMP-mediated process (15). These factors must be considered in attempting to evaluate the physiologic consequences of denervation on the ,&adrenergic response. One finding of potential value of this study relates to the inhibitory action of the catecholamines on skeletal muscle protein catabolism (9). The loss of the p-adrenergic response with denervation that we observed may represent an initial event in the increased protein catabolism with the attendant loss of muscle weight characteristic of the denervated state of skeletal muscle. REFERENCES 1. 2.
3.
4. 5.
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8.
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11.
C. G., AND S. H. APPEL. 1973. Macromolecular characterization of muscle membranes. J. Biol. Chewz. 248 : 5156-5163. ANDREW, C. G., R. R. ALMON, AND S. H. APPEL. 1974. Macromolecular characterization of muscle membranes: Acetylcholine receptor of normal and denervated muscle. J. Biol. Chew 249: 6163-6165. ATLAS, D., M. L. STEER, AND A. LEVITZKI. 1974. Stereospecific binding of propranolol and catecholamines to the p-adrenergic receptor. Proc. Natl. Acad. Sci. U.S.A. 71: 4246-4248. CARLSEN, R. C. 1975. The possible role of cyclic AMP in the neurotrophic control of skeletal muscle. J. Physiol. (London) 247: 343-361. GOLDBERG, A. L., C. JABLECKI, AND J. B. LI. 1947. Effects of use and disuse on amino acid transport and protein turnover in muscle. Ann. N.Y. Acad. Sci. 228: 190-201. GREFRATH, S. P., P. B. SMITH, AND S. H. APPEL. 1976. p-Adrenergic receptor and catecholamine stimulated adenyl cyclase in normal and denervated skeletal muscle sarcolemma. Neurosci. Abstr. 2: 466. GREFRATH, S. P., P. B. SMITH, AND S. H. APPEL. 1977. @-Adrenergic receptor and adenyl cyclase in mammalian muscle sarcolemma. Arch. Biochem. Biophys. (Submitted). GUTMANN, E. 1976. Neurotrophic relations. Anuu. Rev. Physiol. 38 : 177-216. KARL, I. E., A. J. GARBER, AND D. M. KIPNIS. 1976. Alanine and glutamine synthesis and release from skeletal muscle. III. Dietary and hormonal regulation. J. Biol. Chew 251: 844-850. LACOMBE, M. L., E. RENE, G. GUELLAN, AND J. HANOUNE. 1976. Transformation of the & adrenoceptor in normal rat liver into p1 type in Zajdela hepatoma. Nature (London) 262 : 70-72. LEFKOWITZ, R. J., AND M. G. CARON. 1975. Characteristics of S-guanylyl imidodiphosphate activated adenylate cyclase. J. Biol. Chem. 251) : 4418-4422. ANDREW,
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12. LEFKOWITZ, R. J,, L. E. LIMBIRD, C. MUKHERJEE, AND M. G. CARON. 1976.The P-adrenergicreceptorand adenylatecyclase.Biochim.Biopkys. Acta 457: 1-39. 13. LEVITZKI, A., N. SEVILLA, D. ATLAS, AND M. L. STEER. 197.5. Ligand specificity and characteristics of the /3-adrenergic receptor in turkey erythrocyte plasma membranes. J. Mol. Biol. 97: 35-53. 14. LOWRY, 0. H., N. J. ROSEBROLJGH, A. L. FARR, AND R. J. RANDALL. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chcrrt. 193: 265-275. 15. LYNCH, T. J., E. A. TALLANT, AND U. Y. CHEUNY. 1976. Stimulation of rat brain adenylate cyclase by an endogenous protein activator. Fed. Proc. 35: 1633. 16. NARAHARA, H. T., AND C. F. CORI. 1968. Hormonal control of carbohydrate metabolism in muscle. Pages 375-391 in F. DICKENS, P. J. RANDLE, AND W. J. WHELAN, Ed., Carbohydrate Metabolism a?ld Its Disorders Academic Press, New York. 17. RODBELL, M., M. C. LIN, Y. SALOMON, C. LONDOS, J. P. HARWOOD, B. R. MARTIN, M. RENDELL, AND M. BERMAN. 1975. Role of adenine and guanine nucleotides in the activity and response of adenylate cyclase systems to hormones : Evidence for multisite transition states. Pages 3-30 in G. I. DRUMMOND, P. GREENGARD, AND G. A. ROBISON, Ed., Advances in Cyclic Nucleotide Research, Vol. 5. Raven Press, New York. 18. ROSEN, 0. M., AND S. N. ROSEN. 1968. The effect of catecholamines on the adenyl cyclase of frog and tadpole hemolysates. Biochem. Biophys. lies. Commun. 31: 82-91. 19. SALOMON, V., C. LONDOS, AND M. RODBELL. 1974. A highly sensitive adenylate cyclase assay. A?tal. Biochem. 58 : 541-548. 20. SEVILLA, N., M. L. STEER, AND LEVITZKI. 1976. Synergistic activation of adenylate cyclase by guanylyl imidophosphate and epinephrine. Biochemistry 15 : 349334$8. 21. SILZITH, P. B., AND S. H. APPEL. 1976. Biochemical analysis of surface membrane alterations in denervated mammalian muscle. Fed. Proc. 35: 1652. 22. SMITH, P. B., AND S. H. APPEL. 1977. Development of denervation alterations in surface membranes of mammalian skeletal muscle. Exfi. Neurol. 56: 102-114. 23. SMITH, P. B., AND S. H. APPEL. 1977. Isolation and characterization of the surface membranes of fast and slow mammalian skeletal muscle. Biochim. Biophys. Acta 466 : 109-122.