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Application for Research Grant NICOTINE EFFECT UPON CARDIAC' MEMBRANE ENZYMES Harry Darrow Brown The University of Texas Medical Branch at Galveston Galveston, Texas 77550 713 - SO 5 - 1107

Diazacholesterol effect upon membrne ATPase HARRY DARROW BROWN, SWARAJ K. CHATTOPADHYAY and ANIL B. PATEL' :Galveston Texas Conditions resembling myopathy have been induced in experimental animals by the administration of steroids. Response of animals to these compounds has been reported as characteristically a moderate to severe muscular weakness bearing a after contraction, indistinguisha?;le from naturally occurring rrnyotonia. ~ and in the goat reported that the drug induced a delayed muscle fiber relaxation similarity to myotonia.la 2 Winer, et al.3 using 20, 25 diazacholesteroli in man We have found in man and in ;he duck4t 5 that certain, myopathic states can be correlated with an altered respor.se to the cardiac glycoside, ouabain, of rnembrane- skeletal muscle. In the present study we undertook a proionged' administration of bound ATPase from erythrocyte ghosts and! from the sarcoplasmic reticulum of 20, 25 diazacholesterol to rabbits while monitoring the response of isolated erythro- • ATPase preparations was assayed at the termination of the treatment period. cyte-ghosts ATPase to ouabain. Effect of the drug course upon muscle membrane Fifteen male rabbits (6-7 lbs.) were used in three groups. Each animal from Group I was treated with a 5 mg daily dose of 20, 25 diazacholesterol in 1 ml of water daily. The control Group I1'_. was maintained upon a daily administration of of the drug which were progressively increased from 10 mg to 50 mg in 1 mii of water intramuscularly. Similarly, animals from Group II were given higher doses 1 ml of distilled water. From the Biochemistry Departmezt, The University of Texas Medical Branch at Galveston, Texas.

Nine to ten ml of blood was collected in• a glass tube containing 10 ml haemo- lyzing solution ( Tris buffer, 0.002'M, pI-: 7.4, with 0.005 M Na2EDTA ) from each animal. Blood samples were co;ic ::od before the treatment and~ after 7, 8, 1'.' and 13 days of administration. Ske1, <: muscle samples were collected after 16 days of drug therapy. Each time, trc :'•'Iod' haemolyzate was centrifuged at 20, 000 x g for 20 minutes,` The supernatant .:.s discarded and the pellet washed and recen- , : r ..~ trifuged 4 to 5 times in the same ',is buffer, and 2mM NaC1. Before each wash p waer theellets hornognized in a I;•c.xer driven Teflon-pestle homogenizer fo 5 minutes to ensure complete disi,i ion of blood cells. The reddish-white pellet obtained in final centrifugation wa_- collected and stored at -10°C as the erythro- cyte ghost preparation, and used as the source of ATPase activity. Muscles were macerated~ in 10 volumes of 0.1 M cold Tris buffer, pH 7.2, with 0.25 M sucrose preliminary to separation of the membrane fractions by centri- ' fiigation. The slurry was first cerLtrifuged 600 x g for 20 minutes. The pellet was rejected and~ the supernatant dialyzed against the same Tris-sucrose buffer with~ 5 mM Na2EDTA for 4 hours. Afte-=ward the dialysates were centrifuged 10, 000 x g and 20, 000 x g for 30 minutes, and'i each time the pellets were discarded. This supernatant was then centrifuged in 10 ml tubes at 80, 000 x g for 30 minutes and again 100, 000 x g for 70 minutes. The 100, 000 x g pellet was resuspended in 2 ml- Tris-sucrose buffer, pH 7.2, as the enzymatically active fraction ( sarcoplasmic reticulum ) . ATPase activity was measured as inorganic phosphate evolved in reaction mixtures containing 0.1 ml of enzyane preparation (ghost or membrane fraction 0.8 ml substrate ( 0.1 M Tris-sucrose buffer, pH 7.2, with~ Na2ATP r0.3 mg/ml of reaction mixture~ together with 0.001 M MgC12, 0.0021VI KCl, 0.001 M NaCl ), 0.1 ml water or of inhibitor in water. Multiple reactions were run so that they might be taken as samples in sequence to allow consideration of the extent of reaction as a function of time. Reaction mixtures were incubated at 42°. After incubation, i 0.1 ml of 50170 trichloroacetic acid was added and the mixture was centrifuged at ~ 600 x g for 6 minutes. The supernatant was assayed~ for inorganic phosphate. C Protein was determined in sample aliquots of the enzyme preparation. 0 C4 ~ ~ ~

Blood was drawn from each of thc- animals before the initiation of the admin- istration of diazacholesterol .' At this time erythrocyte -ghost ATPase activity tvas inhibited by 10-4.IVf ouabain in the incubation mixture, without exception. ' In the first experimental group, animals receiving 5 mg daily doses, the erythro- cyte adenosine triphosphatase activity was stimulated by ouabain, rather than 'inhibited as were the control preparations. This was true also of preparations from blood of rabbits in experimental Group II, which had received larger doses. These data are presented in Table I. Muscular weakness began the 4th day of drug administrati= and became pro- gressively severe. This was accompanied by an apparent muscle atrophy. Figure 1 A is a graphic representation of the result of a series of experiments in which the catalytic activity was inhibited by 10-41VI' ouabain. Identical experi- in which ATPase activity of ghosts was plotted as a function of time and compared~ (dotted line ) with a similar series, using ghosts prepared' from the same animal, reticulum ) ATPase was similar to that apon the red-blood-cell ATPase; enzyme The results of the drug course upon tInc t00, 000 x g muscle fraction~ ( sarcoplasrnicc effect of ouabain here was to stimLlatc: rather than to inhibit the ATPase activity. ministration of 20, 25 diazacholesterol LLre presented by Figures 1 B and 1 C. The ments using preparations from anirnals which had been carried on courses of ad- stimulated. Data obtained in representative series of experiments is presented in activity of'control animals was inhibited while that of the experimental animals was Figure 2. The characteristic response of membrane ATPase systems to ouabain is an inhibition of the rate of catalytic hydrolysis of ATP. Enzymatic activity of ghost preparations and a muscle merrnbrane fraction from untreated rabbits was inhibited by ouabain. Sustained treatment of animals with 20,25 dYazacholesterol changed the character of the ATPase activity such that a stimulation of the catalytic rate was effected by the presence of ouabain in the incubation mixture. We have

suggestedb, I that the ATPase respons:, to ouabain is a reflection of the conformation of the enzyme which it derives in part from, its association withi the membrane. Myopathic changes in skeletal,muscle following the administration of cortico- steroids has been discussed by Awad, ct al.1- In their experimental work the possibility_of neural induction of the observed myopathy was eliminated and they concluded that steroid myopathy is a primary muscular disorder. .The use of 20,25 diazacholesteroi as a cholesterol lowering agent was discon- tinued in medical, practice when, it became known that the site of action was at the _point of coiiv~rsion of desmosterol to cholesterol resulting in the accumulation of desmosterol. The present results wL'.:.?z show an effect upon membrane related! ion ,transport enzyme (Na+ + K+ -ATPase: may provide an element of support to the : thesis that diazacholesterol causes myatonia by affecting the muscle membrane system. Such an effect could conceiva:)iy follow a change in the lipid pool from which the membrane elements in turn, are synthesized. It is known that the ATPase interrelationship of lipid pool to functional membrane is a matter which requires, activity is affected by changes in the lipid components of the membrane 8 The and is subject to, direct experimental test. Rabbits in the experimental groups visibly showed indications of changes in muscle tone which may relate to the observations of Winer, et al 3 that the diaza- - cholesterol is capable of inducing myotonia. The transport ATPase response to ouabain which we have found in erythrocytes from myotonic patients4 is paralleled port phenomena and appears to indicate that diazacholesterol adrninistration directly affects the membrane transport system. f4id'i.ngs a-ccord _with the common supposition that myotonia involves aberrant trans- by the present experimental results in nhe diazacholesterol-treated rabbit. These 5

Table I'. Effects of ouabain upon a:"' Psse activity# ( µmoles Pi/mg Protein/min ) _ Group II progressively increasing c;oses 10-50 mg/day; Group III, controls.) of erythrocyte ghosts .( Group I a~: :~als received 5 mg diazacholesteroL/day• 6 140 mg _10 Group .roup III. 360 mg 140 mg 360 mg 180 mg 36&mg 360 mg - 310 mg 360 mg Activity Control Ouabain 10-4 M % stimulation inhibition 0.20 0.27 +35 0.22 0.26 +18 0.16 0.17 + 6 0.19 0.21 +10 0.48 0.47 - 2. 0.26 0.39 +50 0.19 0.19 + 0 0.40 0.59 +47 0.35 +52 0.21 0.25 0.39 0.36 0.13 0.15' +15 0.32 0.36 +12 • 0.29 0.46 0.28 0.55 0.24 0.49 +58 +96 + 104 0.15 0.24 +60 0.21 0.32 +52 0.14 0.11 -21 0.16 6.11 -.25 0.14 0.06 -57 0.15 0.12 -20 0.44 ' 0.28 - 36 * Upon the basis of 40 minute incubation.

REFERENCES . Faludi, Georgina, Mills, Lew~is C., and Chayes, Zev W.: Effect of steroids on muscle, Acta Endocrinologica 45: 68-78, 1964. . Awad, Essam A., Swaiman~ Kenneth F., and Kittke, Frederic J.: Changes in the structure, innervation, electromyographic patterns ;` and enzymes of skeletal muscle resultind from experimental treatment with triamcinolone, Arch. Physical Med. and''Rehabilitation 46: 297, 1965 Y } . Winer, Nathaniel, Martt,, Jack M., Somers,' John E., Wolcott, Lester, ° Dale, Homer E., and Burns, Thomas W.: Induced myotonia in man aad goat, J. of Laboratory and Clin. Med. 66: 758-769, 1965. . Brown, H. D., Chattopadhyay, S. K., and Patel, A. B. Erythrocyte ab- normality in human myopatl-: yT . Ms. submitted,. . Brown, H. D., Chattopadr yay. S. K., Patel, A., and Rigdon, R. H.: : Glycoside effect upon n-.enz:;:-:ne enzymes of erythrocytes and muscle in duck myopathy. Expcric_ , ia, in press. . Brown, H. D.: A chasacterizarion of the ouabain sensitivity of heart microsomal ATPase. Bioch. Bioph. Acta 120: 162-165, 1966. . Brown, H. D., Neucere, N. J.,, Altschul, A. M., and Evans, W. J.: Activity patterns of purified ATPase from Arachis, Life Sciences 4: 1439-1447, 1965. • .. . . Schatzmann, H.,J.: Lipoprotein nature of red cell adenosine triphosphatase, Nature 196: 677, 1962. a ,, 1; 1:r 4 rk! .14i'.: 11 " . .. . . . . V .. ~ . ~a I ,

Y:n! r 1s , s. t (Abstzact ) . . . .. _ .. . .. . .. . . - . _ . '. . ..v.. ATPase activity of erythrocytee ghost prer.arations and of a muscle membrane fraction (sarcoplasmic reticulum )-frornl untreated r~.bbits was inhibi'ted by the cardiac glycoside, ouabain.. Sustained treatment of animals with 20,25 diaza- ~. ,_ . . .~., cholesterol, reported by Winer e`t al.,( this Journal, 66: 758, 1965) to in-, " R. ,<duce myotonia, changed the character of the ATPase activity of identical . .. . . ' . . . . :Y: preparations such that a stimulation of the catalytic rate was effected by the . presence of ouabain in the incubation mixture. We have earlier suggested that the ATPase response to ouabain is a reflection of the conformation of the enzyme which it derives in part from its association with the membrane. The 'present findings accord with the common supposition that myotonia involves .`aberrant transport phenomena and~ appears to indicate that` diazacholesterol administration directly affects the membrane transport systemi. • ;sq vr. X! fu .,, w! s s. r x . ... , ._. .., . _ . ., , .. , _ .. - . _ . .