Cyclic amp phosphodiesterase activity in normal and dystrophic human muscle

Cyclic amp phosphodiesterase activity in normal and dystrophic human muscle

435 Clinica Chimica Acta, 50 (1974) 435-437 o Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands SHORT COMMUNICATION CC...

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435

Clinica Chimica Acta, 50 (1974) 435-437 o Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

SHORT COMMUNICATION

CCA 6245

CYCLIC AMP PHOSPHODIESTERASE DYSTROPHIC HUMAN MUSCLE

ACTIVITY

IN NORMAL AND

NIRMAL C. KAR and CARL M. PEARSON Department of Medicine, U.C.L.A. School of Medicine, Los Angeles, Calif. 90024 (U.S.A.) (Received September 14, 1973)

Summary Cyclic AMP phosphodiester~e activity was de~rmined in skeletal muscle of normal subjects and of patients with Duchenne, limb girdle and facioscapulohumeral types of muscular dystrophies and certain neuromuscular disorders. The phosphodiesterase activity levels in the presence of 1 mmole cyclic AMP were unaltered in all diseases that we examined.

There is considerable evidence which indicates that adenosine 3’, 5’-monophosphate (cyclic AMP) acts as an important metabolic regulator in a variety of tissues including skeletal muscle [l]. Several of these cyclic AMP-mediated metabolic processes are shown to be altered in muscle of patients with Duchenne muscular dystrophy (DMD). These include the glycogen phosphorylase system [Z-53, glucose oxidation [2,3] and lipolysis [ 6-81. These findings suggested the possibility of abnormal cyclic AMP metabolism in muscle of DMD patients. Since intracellular concentrations of cyclic AMP are in part determined by the rate of hydrolysis of this nucleotide by a specific phosphodiesterase it occurred to us that the activity of this enzyme may undergo changes in dystrophic muscle. In this report we describe cyclic AMP phosphodiesterase activity in biopsied skeletal muscle tissue of normal subjects and of patients with DMD, limb girdle and facioscapulohumeral types of muscular dystrophies and certain neuromuscular diseases. Muscle specimens were biopsies of gastrocnemius, deltoid or quadriceps obtained from 3 patients with DMD, aged 7-8, one manifesting Duchenne dystrophy female carrier, aged 7, 4 patients with limb girdle dystrophy, aged 10-60, 5 patients with facioscapulohumeral dystrophy, aged 14-45, 4 patients with polymyositis, aged 23-72 and 4 patients with spinal muscular atrophy, aged 10-65. The normal samples of the same muscles were obtained at opera-

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TABLE

I

CYCLIC

AMP

PHOSPHODIESTERASE

ACTIVITY

Disease

OF

HUMAN

None

(4)*

DMD

(3)

*

Manifesting

Duchenne

Limb

dystrophy

girdle

Facioscapulohumeral Polymyositis

* Activity Number

* * * Mean

dystrophy

carrier

(1)

(4) dystrophy

atrophy

(5)

(4)

is expressed of cases

t standard

as nmoles

*

106.0

+

6.76***

115.4

k

9.96

123.6

(4)

muscular

**

HOMOGE:NATES

PhosphodiesterasP activity

Spinal

MUSCLE

of Pi releasedimg

127.6

+ 10.58

95.6

* 13.44

107.6

t

9.26

98.6

t

12.16

of noncollagen

protein

per h at 37O

studied. error.

tion from 4 persons, aged 23-44, who showed no evidence of muscle disease. The preparation of tissue homogenate and the determination of non-collagen protein content of muscle were done as described before [ 91. Cyclic AMP phosphodiesterase activity was determined by the method of Butcher and Sutherland [lo]. The reaction mixture contained 40 mmoles Tris--HCl buffer, pH 7.5, 15 mmoles MgS04 , 1 mmole cyclic AMP (Sigma Chemical Company, St. Louis, U.S.A.) and 0.1 ml muscle homogenate in a final volume of 0.5 ml. After the first 45 min of incubation, 0.1 mg 5’-nucleotidase (from Crotalus adamenteus venom) was added and the mixture was further incubated for 15 min at 37”. The entire reaction was terminated with 0.1 ml of 20% cold HClO, and the released Pi was determined by the method of Fiske and SubbaRow [ll]. For each assay, a blank was prepared by adding the homogenate after incubation. As indicated in Table I, the mean activities of phosphodiesterase in muscle from patients with DMD and with limb girdle or facioscapulohumeral types of muscular dystrophies were not significantly different from those in normal controls. The activity was also unaltered in polymyositis and spinal muscular atrophy. As in the case of adenylate cyclase, which catalyzes the synthesis of cyclic AMP, changes in the activity of phosphodiesterase [l] may be expected to have profound effects on the intracellular concentrations of cyclic AMP. The results of our study did not reveal any significant abnormality in cyclic AMP phosphodiesterase activity in dystrophic muscle when assayed in the presence of 1 mmole cyclic AMP. However, in view of the recent evidence that crude extracts of skeletal muscle [12], like most other tissues, contain two phosphodiesterase activities which are distinguished by widely different affinities for cyclic AMP, further kinetic studies of these enzymes in dystrophic muscle may be needed to entirely rule out any defect in this degradative pathway. Acknowledgments This study was supported Muscular Dystrophy Associations

by U.S.P.H.S. Grant of America, Inc.

GM 15759

and by the

437

References 1 G.A. Robison, R.W. Butcher and E.W. Sutherland, Cyclic AMP, Academic Press, New York, 1971, PP. 1-517. 2 J.C. Dreyfus, G. Schapira and F. Schapira, J. Clin. Invest., 33(1954) 794. 3 P.J. Vignos, Jr. and M. Lefkowitz, J. Clin. Invest., 38 (1959) 873. 4 J.W. Hess. J. Lab. Clin. Med., 66 (1965) 452. 5 S. DiMauro, C. Angelini and C. Catani. J. Neural. Neurosurg. Psychiat., 30 (1967) 411. 6 C.M. Pearson, in G.H. Bourne and M.N. Golarr (Eds). Muscular Dystrophy in Man and Animals, Hafner Publishing Co., New York. p. 1. I C.H. Lin, A.J. Hudson and K.P. Strickland, Life Sci., 11 (1972) 355. 8 N.C. Kar and C.M. Pearson, CIin, Chim. Acta, 38 (1972) 183. 9 N.C. Kar and C.M. Pearson, Clin. Chim. Acta, 40 (1972) 341. 10 R.W. Butcher and E.W. Sutherland, J. Biol. Chem., 237 (1962) 1244. 11 C.H. Fiske and Y. SubbaRow, J. Biol. Chem., 66 (1925) 375. 12 Y.C. Huang and R.G. Kemp, Biochemistry, 10 (1972) 2278.