FABAD J. Pharnı. Sci., 22, 45-49, 1997
RESEARCH ARTICLES I BlLIMSEL ARAŞTIRMALAR
Effects of Taurine Application on the
Prostaglandin and Malondialdehyde Levels in Skeletal Muscle Atrophy Induced by
Denervation
Hale SAY AN*0, Lamia Pımır Y ANIÇOÖLU*
Effects ofTaurine Application on the Prostaglandin and Malondia/dehyde Levels ln Skeletal Muscle Atrophy
lnduced by Denervation
Summary : The ejfects of taurine as an antioxidant and Ca2+
stabiliıer on the denen1ated fast-nı1itch gastrocnernius and slow-tvvitch soleus ınuscles of the rats -..vere investigated.
Transport of taurine İnto skeletal nıuscle w'as peifonned by the intraperitoneal injection of 150mg!kglday taurine, beginning 6
Jıours before neurotoıny and lasting 10 days. After 10 days fo/lowing neurotonıy the aııilnals lvere sacrificed and malondialdelryde (MDA) and prostaglandin E2 like activity (PGLA) levels of both denervated and 11011-treated ınuscles and denervated and taurine-treated muscles were ıneasured.
MDA levels (X±SE)of the denervated and taurine-treated
gastrocneınius muscles were (17.3±2.2 nrnollg) lower tlıan denervated and non-freated controls(67±4.8 nmollg)But there were no significanf differences bet»1een taurine-treated and non-treated soleus rnuscles. Alsa the PGLA levels (X±SE) of denervated and taurine-treated gastrocnemius ınuscles were (24.4±3ng/g) loYı'er than denervated and non-treated controls (39.2±42ng/g); but tlıe PGLA /eve/s of denervated and taurine-treated soleus ınuscles were Jıigher tlıan denervated and non-treated controls (41.1±6.5, 25.2±3nglg respectively).
The protective effects of taurine against lipid peroxidation and PGE2 production in the denervated muscles Yı'ere found to be Tnuch greater in fast-tvvitch gastrocneınius muscles than slow-twitch soleus nıuscles.
Key words: Skeletal ınuscle atrophy, taurine, oxidative stress.
Received Revised Accepted
18.11.1996 15.1.1997 31.1.1997
Introduction
It is well known that differentiation and maintenance of skeletal muscle fibers are intimately regulated by the nerve supply of the muscle1, If skeletal muscle is denervated, the nature and extent of denervation
Denervasyona Bağlı İskelet Kası Atrofisinde Taurin
Uygulamasının Prostaglandin ve Malondialdelıid
Düzeylerine Etkisi
Özet : Bir antioksidan ve Ca2+ stabilizatörü olan ta11ri11i11 raflarda, den.erve hızlı-kasılan gastroknenıius ve yavaş-kasılaıı
soleus kaslarına etkileri incelendi.
Nörotonıiden 6 saat önce başlaytp, 10 gün süre ile günde 150 n1glkg taurin irıtraperitoneal olarak enjekte edilerek; iskelet
kaslarına taurinin transportu gerçekleştirildi. Nörotonıiden
10 gün sonra hayvanlar feda edilerek; tedavi gönneyen denerve kaslar ile taurin-uygulan.nuş denerve kaslarda
nıalondialdehirj (MDA) ve prostagla11di11 E2 benzeri aktivite (PGLA) düzeyleri ölçüldü.
Taurin uygulanan denerve gastroknenıius kaslarında MDA düzeyleri (X±Sl!) (17.3±2.2nınol!g), tedavi uygulaıunanuş
denerve kontrollara göre (67±4.8nnıol!g) düşük bulundu.
Fakat taurin uygulanınış ve uygulannıanuş soleus kaslarında anlanı!t bir fark gözlennıedi. Aynı şekilde tauri11-uygulaıun1ş
denerve gastrokneınius kaslarında PGLA diizeyleri (X±Sll) (24.4±3nglg), tedavi yapıbnamış denerve kontrollara nazaran (39.2±4.2ng!g) düşük idi. Fakat tauri11-uygula111nış sofeus kaslarında PGLA (41.J ±D.5ng/ g), kontrollere nazaran (25.2±3nglg) yüksek bnlundu.
Denenıe kaslarda taurinin lipid peroksidasyonu ve PGE2
üretirnine karşı koruyucu etkisinin, lııılı-kasılan gastroknenıius kaslarında, yavaş-kasılan soleus kaslarrna nazaran daha fazla olduğu saptandı.
atrophy may vary with regard to fiber typel,2. Neu- rotomy in adult animals causes a preferential atrophy of type Il fibers, because these fibers might be more dependent on neural influence than type l fibers2.3, With denervation or disuse, skeletal muscles undergo rapid atropl\y leading to a profound decrease in size,
* Gazi University, Faculty of Medicine, Departınent of Physiology, 06510 Beşevler, Ankara.
Correspondence
Sayan, Yanıçoğlu
protein content and contractile strength4. The pri- mary cause of muscle wasting in denervation atro- phy is the enhancement of protein breakdown. Fol- lowing denervation, the activities of lysosomal protease havc becn reported to increase4. Musde also conlains a large amounı of Ca2•-dependent protease.
Calcium induced an increase in protein breakdown consistent with the structural changes observc>d in musde, and may be linked to a Ca2+ -stimulated re~
lease of lysosomal enzymes and sarcoplasmic pro- teinasesS,6. Also denervation of skeletal muscle caus- es degencrative changes leading to alterations of the sarcolemmal and mitochondrial membrancs7,S,9,ıo.
Thc decreased respiratory ac!ivity found in de- nervated mitochondria is possibly due ıo inncr mem- brane damage caused by Ca2+ induccd swelling. The increased cytosolic Ca2+ concentrations observed could result in Ca2+ release from the sarcoplasmic re- ticulum due possibly lo a deficil of energy ora specif- ic neurotrophic factor in denervated stateH Ca2+ is known to be important in regulating many cellular functions. Deficiencies in normal regulation and in- creasing the amount of free Ca2+ in skeletal muscle may cause some abnormalitics, such as stimulation of protein breakdown!O, leading to increased syn- thesis of PGE2 6 and stimulaıion of lipid per- oxidationl5,16,l7. Rodeman el al. (1982), proposed !hat PGE2 mediates the protein catabolic action of ele- vated cellular Ca2+ in skeletal muscle. According to this concept, increase in cellular concenlration of cal- cium stimulates phospholipase A2, which is Ca2+ de- pendent. This enzyme releases arachidonic acid from membrane phospholipids. This in turn leads to in- creased synthesis of prostaglandins (PGs) that !hen activale !he lysosomal and nonlysosomal enzymes, cathepsin B and D and Ca2+ activatcd neutral pro- lease. Probably, PGE2 promotcs autophagic vacuole formation6,ıo. PGE2 levels of dencrvated and non- treated rnuscles werc found higher in accordance with the dcgree of the degenerative changes12.
Oxygen free radicals cause cellular darnage by in- ducing lipid peroxidation. in pathological states frcc radicals in many tissues are dcrived from xanthine oxidase metabolism13,14. Kondo et al (1993), dem- onstratcd !hal xanthine oxidase activities in atro- phied muscles were significantly higher than in con- lrols and it is known that calcium activated neutral prolease participates in producing xanlhine oxidasc from the xanthine dehydrogenaselS,16,17.
Taurine (a sulphur containing aminoacid) is a non- essenlial aminoacid found in high concentrations in rnuscle, nerve, brain and other organs. Taurine con- centration in skeletal muscle is markedly dependent on fiber type dislribution. It is more abundant in slow oxidative type l fibers than in !he type lI fibers in nor- mal muscles18,l9. Taurine decreases the rale of loss of calcium transport and increases the A TPase activities of sarcoplasmic reticulum20. So il is suggested that it may function as a membrane stabilizcr on sarco- plasmic reticulum. Apart frorn this, it has been shown that taurine has antioxidant effects. As a dirc'Ct anti- oxidant, taurinc sigrıificantly reduces lipid per- oxidation and as an indirect antioxidanl, it acts tosta- bilize the plasrna mcmbrane21,22,23.
Taurine concentration tends to be higher in denerva- tion, rnuscular dystrophy and rnyotonia18,19. lwata and Baba(l 985) found that chronic taurine ad- ministration prcvcnted catabolic changes of fası
twitch muscle after denervation, suggesting a pro- tectivc role of taurine against proteolytic digestion1 9.
The aim of this study was to dctermine the effects of taurine on lipid peroxidation and PGE2 levels, which are cell-damaging agents, in denervatcd gas- trocnemius and soleus muscles.
Malerials and Methods
Both male and fcmale Wistar Albino rnts (200±10 g) were ancsthetized with Nembutal (30 mg/kg, l.P, So- dium Pentobarbital) and denervated in both hind- limbs as follows. Musdes in !he middle third of the length of the thigh were blunlly separatcd and lem segment of the sciatic nerve was excised about 1crn above the popliteal fossa. TI1ere was no bleeding and
!he skin wound was closed with stitches!0,22.
Controls rcceived no treatrnent and werc left in on- going atrophy for ten days. Thc experimenlal group received 150 mg/kg taurine intraperitoneally 24, daily for ten days, begiıming six hours before neurotomy.
Animals were sacrificed following an overdose of Nembutal and al! the gastrocnemius and soleus mus- cles of both controls and cxperimental groups were removcd, cleared of fal; ncrve and connective tissue.
Small tissues sarnples werc immcdialely dissected and laken far MDA and PGE2 levels measurements.
FABAD J. Phamı. Sci., 22, 45-49, 1997
MDA for "Thiobarbituric Acid Reactive Substance"
was assayed by the spectrophotornetric rnethod of Uchiyarna and Mihara25 . Tissue samples were ho- mogenized in ice-cold % 1.15 KCI. After centrifuga- tion, the supernatant was added to 1 %phosphoric acid and %0,6 thiobarbituric acid. Then mixture is heated for 45 rninutes water bath. Then n-butanol is added and centrifugated. The n-butanol layer is taken for spectrophotometric measurement at 535nm and 520 nm excitation25.
Prostaglandin E2 Like Activity (PGLA) of both the gastrocnemius and the soleus rnuscles was measured by bioassay methods of Gillrnore and Vane26. Two sets of assay tissues tha! were a rat stornach strip and rnuscle extracts were prepared and superfused in polygraph channel for recording thc activation pros- taglandin E2. The small muscle samples were acid- ified with hydrochloric acid and the PG5 in them were extracted with cthyl acetate. TI1e responses to the standard PGE2 of the s!omach strips compared with the responses to sample extracts. The changes in length of the assay tissues were detected by strain gauges attached to auxotomic levcrs and were re- corded on the Grass 7G model of polygraph.
Statistical analysis: Dala shown in tables and graph- ics are typical of results obtained in at least two in- dependent experiments, in the statistical analyses,
"The Mam1-Whitney U Test'' was used for paired data, based on means ±SE of at lcast six animals.
Results
We compared the atrophied soleus and muscles with the gastrocnemius muscles of each rat in the denervated non-treated controls with the muscles of denervated and taurine-treated experimental groups.
MDA levels in the denervated gastrocnemius and so- leus muscles of controls and taurine- treated expcri- mental groups are shown in the Figure 1. By ten days after neurotomy the mean MDA !eve! in the gas- trocnemius muscles of taurine trealed group was sig- nificantly lower than the gastrocnemius muscles of non-treated controls (p<0.05). But there were no sig- nificant differences between the MDA levels in so- leus muscles of taurine treated and non-treated groups (p>0.05).
:lB 3:t2 l ~O± 4.8 6i±4 8 ll.:li:.2
nmr:-1 /q
Figure 1. MDA Jevels (X±SE) of denervated gas- trocnemius and soleus musclcs of taurine - treat- ed experimental group (n=6) and non-treated controls (n~6); *p>0.05.
Figure 2 co.mpare the PGLA of denervated gas- trocnemius and soleus muscles of taurine treated ex- perimental group and non-treated controls. Levels of PGLA in the gastrocnernius muscles of taurine treat- ed groups were found lower than non-trealed con- trols. The difference was statistically significant
il')/~
Figure 2. PGLA levels (X±SE) of denervated gas- trocnemius and soleus muscles of taurine-treated experimental group (n=6) and non-treated con- trols (n=6); *p>0.05.
(p<0.05). ln.the soleus muscles of taurine-treated ex- perimental group there was an increase in PGLA lev- els compared with the non-treated controls (p<0.05).
Discussion
MDA and PGLA levels have been found high in de- nervated and non-treated gastrocnemius muscles ol controls, but not in soleus musdes. Because it was known that their synthesis increase in skeletal mus- cles due to pathological states such as atrophy and dystrophy; our findings were in accordance with pre- vious reports that denervation caused a preferential atrophy of type Il fibersl,3.
Sayan, Yanıçoğlıı
Physiologically, slow reacting red muscles contain more taurine than fast reacting white muscles18 .
However lwata and Baba showcd that denervation of skeletal muscles increased the transport and the con- tent of taurine in fası twitch muscles but not in slow twitch muscles. They injected tracer amounts of 3(H) taurine intrapcritoneally ıo rats and showed ıha! its transport into fast twitch skeletal muscles seemcd to be time-dependent and reached a maximum at about six hours after the injcction, and the increase of tau- rine in denervated muscle was restricted preferential- ly to fastmuscles rathcr than to slow musclcs19.
in our study it was found that, gastrocnemius mus- cles in the denervated and taurine-treated experi- mental group had lower PGLA and MDA levels.
These findings encouraged us to suggest the pro- tective and antioxidant effects of taurine in pro- teolytic digestion of atrophied muscles.
Taurine enhances the capacity lor calcium uptake by sarcoplasmic reticulum. Thus il is likely that calcium fluxes in skeletal muscle could be regulated or mod- ified by taurine2°. Taurine has been shown to protcct the guinea pig heart against hypoxic and reoxy- genation damage and to attenuate Ca2+ influx during ischemia in the rabbit brain27. Also taurine ad- ministration to patients with dystrophy can mark- edly reduce the electrical signs of myotonia 28,29.
it was repork.'<i that the incrcase in ion and water permeability due to the membrane damage caused by lipid peroxidation was prevented by taurinc probably with a calcium dependent mechanism and in this way it stabilized the membrane23 . According to another re- port it was shown that, tissue MDA content was di- minished in the taurine treated rats 22,23,30_
in our study the effect of taurine on lipid per- oxidation in atrophied skeletal muscle by denerva- tion was determined by MDA production, and we found !hat taurine decreased the MDA levels in gas- trocnemius muscles of taurine-treated group, but not in soleus muscles. Because of the decreased level of lipid peroxidation in gastrocnemius muscles of tau- rine-treated group we suggest that !his aminoacid ex- erts its beneficial effect by acting as an antioxidant.
We observed that the PGLA levels in the gas- trocnemius muscles of taurine-treated expcrimental
groups were lower than non-treated controls. in con- trast, the PGLA levels in the soleus muscles of tau- rine-treated expcrimental groups were a little higher than the non-treated group. Although we expected that in s]ovv-twitch muscles, taurine did not excrt a bcneficial effect, perhaps because these denervated muscles could not reserve enough taurinc to be ef- fective as mentioned by lwata and Baba previously19;
the high PGLA levels of the experimental soleus mus- cles must be investiga ted in forward studics.
The present study demonstrated the possible pro- tective effc"Cts of taurine in the oxidativc strcss and in PGE2 stimulated protein breakdown of fast-twitch muscles following denervation. We suggest !hat tau- rine plays an important role in the protcction of cells
!rom Ca2+ dcpendent proteolytic activity and ox- idative damage by stabilizing the cellular mcmbrane and regulating intracellular Ca2+ concentration in denervation.
This research was supported by Gazi University Re- search Foundation, 11/96-17.
REFERENCES
1) Tomanek RJ,Lund DD. Degeneration of different types of skeletal muscle fibers I. Degeneration, J.!lnat.,116(3), 395-407, 1992.
2) Bajusz E. Red skeletal ınuscle fibers : Relative independence of neural control, Scieııce,145,938-939, 1%4.
3) Jaweed MM, Herbison G, Ditunno J. Denervation and reinnervation of fast and slow musclcs. A histochemical study in rats, f. Histoclıenı. Cytoclıe11ı., 23(11),808-827, 1975.
4) Furuno K, Goodman MN, Goldberg AL. Role of different proteolytic systems in the degradation of muscle proteins during denervation atrophy, f. Biol.
Clıenı., 265(15), 8550-8557,1990.
5) Lewis SEM, Anderson P, Goldspink DF. The effects of calcium on protein turnover in skelctal n1uscle of rat, J.Bioclıeııı., 204, 257-264, 1982.
6) Rodemann HP,Waxman L, Goldberg AL. The stimulation of protein degradation in muscle by Ca2+
is mediated by prostaglandin E2 and docs not require the calcium-activated protcasc, ].Biol. Clıeııı., 257(15), 8716-8723, 1982.
7) Cheah MA. Effect of long chain unsaturated fatty acids on the calcium transport of sarcoplasmic reticulum, Hioclıenı. Biophy.Acta., 648,113-119,1981.
8) jaweed MM, Alam ), Herbison Gj, Ditunno JF.
Prostaglandins in denervated skeletal n1uscle of rat:
Effect ofdirect electrical stimulation, Neuroscience, 6 (12), 2787-2792, 1981.
FABAD J. Pharnı. Sci, 22, 45-49, 1997
9) Tate AC, Bick RJ, Myers TD, Pitts BJ, Van Winkle BW. Alteration of sarcoplasmic reticulum after denervation of chicken pectoralis major ınuscle, Bioclıem. J., 210, 339-344,1983.
10) Turinsky j. Phospholipids, prostaglandin E2 and proteolysis in dencrvated muscle, A11ı.J. Plıysiol., 251, R165-R173,l986.
11) Joffe M, Savage N, Isaacs H. lncreased muscle Ca2+,
Bioclıerıı.J., 196, 663-667, 1981.
12) Yanıçoğlu LP, Babül A, Gönül B, Erbaş D. Effect of prostaglandin- inhibition, electrical stimulation and local heating on skclctal muscle atrophy, f.lslanıic
Acnd. Sci., 2, 216-221, 1993.
13) Grace PA. Ischeınia-reperfusion injury, Br. J. Sıırg.,
81, 637-647, 1986.
14) Freen1an BA, Crapo JD. Biology of disease free radicals and tissue injury, Lab. fııvest., 45(5), 417-426, 1982.
15) Kondo H, Miura M, Itokov.'a Y. Oxidative stress in skeletal rnusclc atrophied by immobilization, Acta
Plıysiol. Scand., 142, 527-528, 1991.
16) Kondo H, Miura M, Kodeme ), Ahmed SM, Hokowa Y. Role of iron in oxidative stress in skeletal muscle atrophied by immobilization, Pfliiger Arc., 421, 295-297, 1992.
17) Kondo H, Miura M, Nakagaki 1, Sasaki S, Itokowa Y.
Mechanism of oxidative stress in skeletal muscle atrophied by immobilization, Anı. f.Physiol., 265(28), E839-E844, 1993.
18) Airaksinen EM, Paljarvi L, Partanen, J, Collan Y, Leakso R, Pentikainen T. Taurine in normal and discased human skeletal muscle, Acta Neurol. Scand., 81,1-7, 1990.
19) Iwata H, Baba A. Spesific increase of taurinc in denervated skeletal muscle, Taurine: Biological actions and clinical perspectives, Alan R Liss Inc., pp.397-405,1985.
20) Huxtable R, Bressler R. Effect of taurine on a muscle intracellular n1embrane, Bioclzinz.Biophys. Acta, 323, 573-583, 1973.
21) Banks MA, Parter DW, Martin WG, Castronowa V.
Taurinc protects against oxidant injury to rat alveolar
pneuınocytes, Taurine, Lombardini JB(eds), Plenuın
Press, New York, pp. 341-354, 1992.
22) Tractman H, Pizzo RD, Fulter WS, Levine D, Rao PS.
T aurine attenuates renal disease in chronic puromycin aminonucleoside nephropathy, Anı. J.
Plıysiol., 31, F117-Fl23, 1992.
23) Huxtable R. Physiological actions of taurine, Plıysiol.
Reı•., 72(1), 101-163, 1992.
24) Can1erino DC, Luca AD, Mambrini M, Ferrannini E, Franconi F. The effect of taurine on pharmacologically induced ınyotonia, Niuscle aııd
Ncrve, 12, 898-904, 1989.
25) Uchiyama M, Mihara M. Dctermination of malondialdehydc prccursor in tissues by thiobarbituric acid test, Anıı. Bioclıe111., 86, 271- 278, 1978.
26) Gillrnore N, Vane SR, Wyllie SH. Prostaglandin release by spleen, Nat11re, 218, 1135-1140, 1968.
27) Schurr A, Tseng MT, West CA, Rigor BM. Taurine improves the recovery of neuronal function following cerebral ischemia: An invitro study, Life Scie11ce, 40, 2059-2066, 1987.
28) Dinçer S,Babül A, Erdoğan D, Özoğul C, Dinçer SL.
Effect of taurine on wound healing, Anıinoacids,
10,59-71,1996.
29) Duelli L, Mutani R, Fassio F. The treatment of myotonia: Evaluation of chronic oral taurine therapy,
Neıırology, 33, 599-603, 1983.
30) Iwata H, Kim KB, Fukiu Y, Baba A. Ne11ral regulation of taıırine transport in skeletal rnııscle, Taııri11e in lıealtlı
a11d disease, Huxtable R, Michalk DV(eds), Plenurn Press, New York, pp.158-163, 1994.
