Phospholipid distribution and stimulation of methylation during denervation and reinnervation in skeletal muscle

Neuroehemistry International. Vol. 5, No. 6, pp. 763 771. 1983 Printed in Great Britain. All rights reserved

0197-0186/83-$3.00+0.00 ',L'I 1983 Pergamon Press Ltd

P H O S P H O L I P I D D I S T R I B U T I O N A N D S T I M U L A T I O N OF METHYLATION DURING DENERVATION AND R E I N N E R V A T I O N IN SKELETAL M U S C L E PHILIP ROSENBERG,* ROBERT E. PANNI* and WOLF D. DETTBARNt *Section of Pharmacology and Toxicology, School of Pharmacy, The University of Connecticut, Storrs, CT 06268 and tDepartment of Pharmacology, Vanderbilt University, Nashville, TN 3?232, U.S.A. (Received 11 January 1983; accepted 28 March 1983)

Abstraet--Denervation of the rat soleus and extensor digitorum longus muscles was induced by nerve crush. Functional signs of denervation were noted within 48 h with recovery beginning about the 12th day following denervation. There was also a marked decrease in muscle weight but only a small decrease in protein content per mg of muscle, subsequent to denervation. At 1, 2 and 3 weeks following nerve crush there was a relative decrease in muscle phosphatidylethanolamine (PE) and a corresponding increase in phosphatidylcholine (PC). The proportion of the other phospholipids did not significantly change. The levels of PC and PE returned to, or in some cases slightly overshot, control values at 4 and 5 weeks following nerve crush, i.e. during the period of reinnervation. Levels in non-denervated contralateral muscles did not significantly change. At 1 and 3 weeks following nerve crush a marked increase was observed in the activities of the enzymes PE-methyltransferases I and II, as measured by [3H]methyl group incorporation from S-adenosyl methionine into phosphatidylmonomethylethanolamine, phosphatidyldimethylethanolamine and PC. Increased activity of these methylases was seen in the contralateral control muscle, although less than in the denervated muscle. These enzymatic changes could be responsible for the changes in PE and PC distribution which we observed. Methylation of PE might also decrease the microviscosity of the membrane, thereby leading to other changes associated with denervation. Activation of this system might be another form of supersensitivity induced by denervation.

Since denervated muscles can be used as model systems in the study of certain neuromuscular disorders, there has been great interest in analyzing those biochemical and physiological changes which occur as a result of denervation. Due to the extensive variety of changes observed following denervation, it has been difficult to evaluate their significance and determine which are the primary effects directly attributable to denervation. The following are some of the major changes observed as a consequence of denervation of skeletal muscle hypersensitivity to acetylcholine and increased numbers of acetylcholine receptors along the entire length of the denervated muscle (Thesleft, 1974; Albuquerque and McIsaac, 1976; Tipnis and Malhutra, 1980), decreased or increased acetylcholinesterase activity dependent upon the species (Eranko and Terfiv~inen, 1967; T e n n y s o n et al., 1977; Davey and Younkin, 1978), nerve sprouting (Hoffman and Springell, 1951; Tweedle and Kabara, 1977), modifications of electrophysiological properties including resting m e m b r a n e and action and junctional potentials (Albuquerque and Thesleft, 1968; Harris, 1974; Albu-

querque et al., 1971 ; McArdle et al., 1980), changes in enzymes of carbohydrate and protein metabolism (Hogan et al., 1965; Wallis and Koenig, 1980; Nemeth et al., 1980) and modifications of the ATPase and adenylate cyclase systems (Festoft et al., 1977a, b; M a r g r e t h et al., 1972). Of special interest in relationship to this study are the reported changes in muscle phospholipids. In addition to maintenance of structural and permeability properties of cellular membranes, phospholipids are also implicated as essential cofactors of membraneb o u n d enzymes and as mediators for various receptor functions (Fourcans and Jain, 1974; Michell, 1975; Rosenberg, 1976; Trudell, 1977; H a w t h o r n e and Pickard, 1979; Loh and Law, 1980; Van Deenen, 1981). Denervation has been associated not only with alterations in phospholipid metabolism but also with a selective decrease in phosphatidylethanolamine (PE) and an increase in phosphatidylcholine (PC) with an overall increase in the concentration of phospholipids (Graft et al., 1965a, b,c; Heiner et al., 1971, 1975; Appel, 1974; Fernandez et al., 1979; K a b a r a and 763

764

PIIII II, Rosl NI'¢I:I>-.(;dl dL

Tweedle. 19;41). T h e s e c h a n g e s m a ; bc parlicularl.~ r e l e \ a n t to n e u r o m u s c u l a r diseases in w h i c h d i s o r d e r s or p h o s p h o l i p i d s h a v e been implicated I K w o k <'# a/.. 1976: D e k r e t s e r a n d Livett, 19771. T h e c h a n g e s i n d u c e d b3 d e n e r x a t i o n on P C a n d PE lcxels c o u l d be m e d i a t e d t h r o u g h an a c t i v a t i o n of the PE m e t h y l t r a n s f e r a s e e n z b m e s y s t e m (S-adenossl-l,methionine: phosphatidylethanolamine ,\"-methyltransferase: Et" 2.1.1.17) w h i c h c o u l d thor1, result iil. a decrease in PE a n d increase in PC levels. T h i s s y s t e m which h a s been extensively s t u d i e d by A x e l r o d a n d c m ~ o r k e r s (Hirata a n d A x e h o d . 1978a. b. 1980. 1982; H i r a t a ct al.. 1978, 1979a. b. 1980: ('rcx~s cl a/.. 1980a. b. cl was first discovered in rat liver ( B r e m c r a n d G r c e n b c r g , 1960: A r t o m a n d L o f l a n d ,h,, 1960} ~ h c r c its properties have been s t u d i e d in c o n s i d e r a b l e detail IArtom. 1964: S c h n e i d e r a n d Vance. 19791. T h i s m e l h x l t r a n s f e r a s c s \ s t e m is also f o u n d in brain I m o z z i a n d Porcellati, 1979: Bhisztajn ct al.. I979: ( r c w s el a/.. 198()a,b: F o n l u p i vt a/.. 1981. 19821. adremil g l a n d I H i r a t a c t a / . , 1978i, red cells a n d m a s t cells (Hirata a n d Axelrod, 1978a: H i r a t a c't a/.. 197K 1979a). plalelets, b a s o p h i l s a n d n e u t r o p h i l s ( H i r a t a <'t al.. 1979b. 1981): C r e w s v t a / . , 1980c: H o t c h k i s s ct a/.. 1981}. a n d in heart m u s c l e ( B r e m e r a n d G r e e n b e r g , 1961: M o g e l s o n a n d Sobel. 19Sli a n d is s t i m u l a t c d as a result of b m d i n g to the cell surface of a n u m b e r of t r a n s m i t t e r s , peptides, lectins etc. M e t h y l a t i o n of PE lcads to mcrcascd inenlbranc iluidity ( H i r a t a and A x e h o d . 197Sbl w h i c h possibly e x p l a i n s m a n } o[ thc apparentl> u n r e l a t e d c h a n g e s m m e m b r a n e properties s u b s e q u e n t to denervation. T h e p u r p o s e s of this s t u d y were to see if u n d e r o u r c o n d i t i o n s \~e o b s e r v e d a r e d i s t r i b u t i o n of the relative p r o p o r t i o n s of P C a n d PE in d e n e r v a t e d m u s c l e a n d if so. to scc if there is an a s s o c i a t e d a c t i x a t i o n of tile p h o s p h o l i p i d m e t h s l a t i o n p a t h w a y w h i c h could explain these lindings. Since m e t h y l l r a n s f e r a s e activity is present in m a n y tissues it s e e m e d likely that the s v s l e m m i g h t also be p r e s e n t in skeletal musclc. Wc selected for lhese studies the rat soleus a n d e x t e n s o r d i g i t o r u m l o n g u s m u s c l e s as e x a m p l e s respectively of slov, a n d fast t~xitch inuscles which h a v e also been tiscd extensively in p r e v i o u s studies on the propcrtics of d e n e r x a t e d muscles.

EXPERIMI:NTAL PRO('EDI. RkS Male, Harlan Sprague Dawlc5 rats (150 200gl ',',,ere kept under identical environmental conditions (temperaturc, light dark cycle etc.) and all received a uniform controlled diet. Rats ,acre anesthetized with ether and the left sciatic nerve was crushed tit tile sciatic notch ovcr a 3 m m s e g m e n t for 30 s a i i h a serrated h e m o s i a t . G r o u p s of live

/lilinlals ',hCle operated u p o n LII OllC thnc, h,,)wcver not till groups (1 week. 2 weeks etc, sec belong) ,acre operated o,1 the samc da>. Postoperativcl 3, the rats '~;ere observed for function of the leg c',er~, other da3. On pulling the rat gentl> b', the tail on a hard surface, the innervated leg extends and tile cla~s grasp al the surface while the dener~ated leg drav, s limpb. As the nerve regenerates to the muscle, return to n o r n l t d ft.inction i,', c v M e n t a n d easib observed The rats ,,,,'ere killed b3 decapitation ;it 1. 2. 3. 4 and 5 ~',ecks after nerve crush. Soleus and cxten:,or digitortlnl longus muscles, I~oth denerxated and contralatera] control. '~erc removed and weighed. Prior lo removing the muscle, tile loss of e n e r v a t i o n or the progress of reinner,,alion v, as tested hx electrical stimulation of tile sciatic nerxc abo'+e and b e h m tllc site of tile crush and the effect oil the muscles was observed.

Phosldlolil,id mca~m'cmcm, Muscles v, crc extracted v, ith chloroform nicihanol {2:1. xl and ~ashcd as described by Folch ~,t a/. (1957). The phospholipids m the cxtracl '~\ere separated using a twodinlensional the-laver chromatographic technique {('oi1drea <,/ a/.. 19671. Following identification with iodine xapor and mnhbdrin reagent tile spols v~erc scraped and lipid phosphorus delcrmmed according to the spectrophotonlclric method of Bartlett ll9591. Thc pcrceni dislrihution o[" each phospholipid rclali\e to thc Iotal phospholipid coiltenl ",',,ascalctllalcd 17}1each samplc. PhoslUmtidvh'Ihamdaminc

#m'thlllransf{'ra.~c~

The meth~,lase s>stenl ,,',as assa,,cd as prc~iousl 5 dcscrihcd IHirata cl a/.. 19781 I*,y measurhlg tile incorporation of tritiatcd mcih 31 groups from S-adenosyl-i -[MetlDi-3H ]melhioninc into the muscle pilospholJpids. All restihs arc presented as p e e l n-lethal incorporaled per 100nlg ,act ~eight of muscle per h. In cxperiments wilh control tissue it ",aas dcnlonslrated lhal the assay \,,tls linear with time 130 t2tlminl and with cunotinl of tissuc {25 100rag ~vct ~wighll. h l c t h l hran.<:ti'ra.,c ctl_-lmc I

The tissues v, crc ]lomogenized ill till tiqtleOi.iS solution containing IOmM Mg('l_,, 0.1 mM sodium EDTA and 5()mM sodium acetatc btif'i'ci (pH 6.5L Aliquots of each muscle homogemite (0.5el containing 50 100mg wet weight tissuci x,,ere transferred to two retiction tubes. To each lubc was added 0. I m g of phosphatidylethanolanmlc and sufficient S-adenosxl methionme to yield a final concentration of 4HM. To one of the tubes. S-adenosyl-l.homocysteine {5 × 10 "*M final concentralion) was added as a specific inhibitor of the S-adenosyl methionine stimulated methyhltion IHirata <'l a/.. 197S: Bhlsztajn ct al., 1979: Fonlupl ctal.. 1981; Mogelson and Sobel. 19Sll. Thc tuhcs v, ere warmed to 3"/ (' and about 50.000dpm of tritiated S-adenosyl mcthioninc was added to each tube. The tubes v, ere incubated tit 37 (" t\w 60 rain and ihe reaction stopped by the addition of I ml of I0",, trichkmtcctic acid. "File phospholipids wcrc exlractcd with chloroform methanol m i x l u r c s l l : 3 : I:1: 2:1. b> xol.){Marinetti el al.. 1959} and sepm'ated on a one-dimensional thin-layer chromatographic s+xstem niilizmg n-propanol propionic acid chloroform water (2:2:1: 1. b', vol.I as the solvent system. Spots Iphosphatid31cthanolanline (P[!i: phosphatidyl monomethyl cthanolanlinc ( PM El: phosphatidyl dimethyl ethanolaminc ( P D I ) : phosphaiid31choline (P('I. wcrc

Methyltransferases in denervated muscle identified by iodine vapor and ninhydrin and compared with known standards. Each spot was scraped onto a filter paper and eluted, into a counting vial, with chloroform methanol (2: 1, v/v) in order to extract the phospholipids. These washings were evaporated to dryness under a stream of nitrogen and the residue redissolved in a toluene PPO (4 g/l) and P O P O P (0.1 g/l) mixture. Tubes were counted in a Beckman LSS000 liquid scintillation cotmter, with all counts being corrected for background. The dpm values of the phospholipid spots (except for PE) were all significantly above background ranging from 300 to l l,000dpm per 100 mg muscle per h depending on methyltransferase activity.

Methyltramferase enzyme II The activity of the second methyltransferase enzyme was assayed similarly as described for enzyme I except for the following modifications. The muscles were homogenized in an aqueous solution of 5 0 m M sodium borate buffer (pH 10) in the absence of magnesium. Instead of PE, 100/~g of phosphatidyl-N-monomethyl ethanolamine was added to each tube and the concentration of S-adenosyl methionine was 200 ItM instead of 4 llM.

Materials Adenosyl-L-methionine, S-[Methyl-3H] contained, dependent upon batch, 55-65 Ci/mmol and was obtained from New England Nuclear (Boston, MA). S-adenosyl-Lmethionine iodide and S-adenosyl-L-homocysteine and phosphatidylethanolamine (dipalmitoyl) were obtained from Sigma Chem. Co., St. Louis, MO. L-phosphatidylN.N-dimethyl ethanolamine and phosphatidyl-N-monomethyl ethanolamine were obtained from Calbiochem Behring Corp., LaJollm CA and GIBCO Laboratories, Grand Island, New York.

765 RESULTS

Function, weiyht and protein content After crush of the sciatic nerve in the m i d - t h i g h region, t r a n s m i s s i o n to the muscles was lost within 24~48 h. Twitch responses of the muscles as a result of indirect stimulation a b o v e the crush site began to a p p e a r between the 12th a n d 14th day. Recovery of leg function in the live animal was o b v i o u s between the third and fourth week while five weeks after nerve crush no significant difference from c o n t r o l was apparent. Muscle twitch height in response to indirect stimulation reached 50°,~i of control level by the end of the fifth week. Decreases in weight and protein c o n t e n t of denervated muscle are s h o w n in Table 1. These c h a n g e s were m u c h greater in the soleus muscle t h a n in the extensor d i g i t o r u m longus muscle. F o r both muscles m a x i m a l weight loss was seen 14 days after nerve crush, at which time the weights were 32 50'!i; o f the c o r r e s p o n d i n g contralateral c o n t r o l muscle. Five weeks after d e n e r v a t i o n the weights o f the muscles had recovered to 65~,; o f the c o n t r o l value. Protein c o n t e n t per m g of muscle decreased no m o r e than 20!~, within 3 weeks following d e n e r v a t i o n a n d regained c o n t r o l values by five weeks.

Phospholipid distribution At 1, 2 and 3 weeks following denervation, there was a significant decrease (P < 0.051 of P E (expressed

Table 1. Effects of denervation on weight and protein content of soleus and extensor digitorum longus muscles Weeks following denervation 0

1

2

3

4

5

156 + 2 151 ± 3

I35 ± 3* 149 _+ 1

134 _+ 3* 162 _+ 7

125 _+ 3* 159 _+ 6

154 _+ 4* 177 _+ 6

163 _+ 4 172 _+ 7

46 ± 5* 90 _+ 6

30 + 1' 94 _+ 4

45 _+ 3* 102 _+ 1

78 _+ 5* 134 _+ 14

84 _+ 5* 132 _+ 11

143 _+ 2* 153 _+ 4

147 + 5* 168 _+ 7

143 + 4* 158 _+ 12

164 _+ 4 162 _+ 10

168 ± 6 166 _+ 11

81 _+ 6 127 _+ 10

65 + 2* 128 _+ 11

79 _+ 3* 138 _+ 5

104 _+ 5* 144 _+ 13

105 ± 5* 158 _+ 11

Solcus Protein (l~g/mg muscle) Denervated left muscle Contralateral control muscle Weight of muscle (mg) Denervated left muscle Contralateral control muscle

80 _+ 10+

Extensor di(Jitorum Ion.qus Protein (pg/mg muscle) Denervated left muscle Contralateral control muscle Weight of muscle (mg) Denervated left muscle Contralateral control muscle

152 _+ 4 153 _+ 2 96 _+ 11+

All results are shown as means _+ S.E. (N = 7). 0 week represents control value prior to denervation: protein was determined by the method of Lowry et al. (1951): *P < 0.01 compared to corresponding contralateral control muscle (Students t-test): +Values for pooled left and right muscles.

766

PltlLIP

SOLEUS

ROSENBJiRG

{'l ~tl.

8.1); phosphatidic

MUSCLE

acid + cardiolipin

(3.5 lO.O).

There were no significant dittcrences in the ",, distribution of the phospholipids m the two muscles.

6O

Pho.w~hafidvh'Hla,u~hu~im' mcHu'/n'an.~l~'rase acdHtr 5O

% 40

,c F I i

o L

....

Con

-

D e n e r vatecJ

-

ol

i

I

l

o

~

2

L

L

d

4

5

WEEKS

Fig. I. Percent distribution of phosphatidylcholine (P('I and phosphatidylethanolamine {PEI Following denervation of the rat soleus muscle. O O and • • = dencrrated left muscle; O O and • • = contralateral non-denervated right muscle: O = P(' values: • = PE values. Abscissa = weeks following denervation (0 = control non-denerwlted value): ordinate = ",, of the total phospholipids in lipid extract from muscle. Results are shown as means + S.E.. each based upon 3 to 4 determinations on different muscles. Asterisks indicate values of denerw~ted muscles which are significantly different IP < 0.05) both from their ov,'n 0 week value and from lhe contralateral trighH muscle of the corresponding week.

as ", of total phospholipids), in soleus and extensor digitorum longus muscles, as compared either to 0 week control value or corresponding right side. nondenervated value (Figs 1, 2). By the fourth week the levels of PE were similar to each other in the denervated left and non-denervated right-side muscles. On the fifth week. there was a tendency towards reversal such that the '!,, PE values were larger in the denervated than in the innervated muscle. Phosphatidylcholine (PC) levels increased following denervation. so that in the denerwlted soleus muscles the o of PC at 1.2 and 3 weeks following denervation was significantly greater than both the 0 week control level and the corresponding '!; value for the right side nondenerwtted muscle. In the extensor digitorum longus the differences were only significant at the three-week interval. The !!,, distribution of the other phospholipids in the muscle extract did not significantly change during the 5-week period. The ranges of values C,, of total phospholipidst over the 5 wccks. including both muscles, were as follows: lysophosphatidylserinc (0.2 2.0t: phosphatidylserinc (2.7 6.0);

sphingomyelin (1.7-4.9); phosphatidylinositol (5.#

Radioactivil> was nlcasured ill the chromatographic spots corresponding to P('. phosphatidyldimethylethanolamine (PD[!). phosphatid,,.hnonomethylethanolamine (PME). and PE. both in thc absence and in the presence of the inhibitor S-adenosylhomocysteme (S-AH). No radioactivity, abovc background, was observed in the P[! spot which is as expected smce PE has no methyl groups. Radioactivity in the spots m the absence of S-AH minus the radioactivity in the presence of S-AH was taken as the measure of lhe specific activity of lhc phosphatidylcthanolamine methvhransferase enz,~mes 1 and 11. The number of counts in the absencc of the inhibitor were usually tx~o to fnur fold greater than in tile presence of the inhibitor. When levels of radioactive incorporation were Vel% low. hov
70

EXTENSOR DIGITORUM LONGUS MUSCLE

60

50

% 40

3O

20

I0

. . . . -

0

(

o

II

Conlrol Denervated

-

---J~ .....

~

2

&

3

4

_ _ j

5

¢4EEKS

Fig. 2. Percent distribution of phosphatidylcholmc (P(I and phosphatidylethanolaminc (PEI l\~llowing denervation of the rat extensor digitorum longus muscle. See Fig. 1 for further details.

Methyltransferases in denervated muscle SOLEUS

037~-

DISCUSSION

MUSCLE

002025:[ METHYL TRIANSFERASE

OOS

025

g

MUSCLE WE~K

Pc-

C~

Lfl

~

12

.~c~,

PL

0

t, DE I .c

METHYL

DC S

DC 0

oc

pIc

DC

DC

DC S

o P'__~E

TRANSFERASE

!!

~

oc

OC DC I 3 PO~

%

o

, POE

767

~ PME

S

Fig. 3. Methyl group incorporation into phospho]ipids following denervation of rat soleus muscle. Incorporation was calculated from the difference in radioactivity in the absence and in the presence of the inhibitor S-adenosyl homocysteine and is expressed as picomoles per 100rag wet weight of muscle per hour. See methods for details of assays for methyl transferase enzymes 1 and II. Abbreviations: D = denervated left muscle (0 week is control left muscle): C = contralateral control right muscle: 0, 1, 3 = weeks following denervation; PC = phosphatidylcholine: PDE = phosphatidyldimethylethanolaminc: PME = phosphatidylmonomethylethanolamine: PL ~ phospholipid. All values are the means of two or three determinations on separate muscles. The standard errors of the means both in the presence and absence of S-adenosyl homocysteine were between 10 and 20"},, of the mean value.

We confirmed previous findings (Appel et al., 1974; Fernandez et al., 1979; K a b a r a and Tweedle, 1981) that subsequent to denervation (Figs I and 2 at 1, 2 and 3 weeks) there is a relative decrease in PE and increase in PC. The magnitude of the changes is also similar to those in the literature. Changes in the contralateral muscles were much less than in the denervated muscles. At 4 and 5 weeks, when reinnervation subsequent to the nerve crush would have occurred, the °~i distribution values for PC and PE returned towards normal. There was also a tendency towards overshoot at 5 weeks; for example in the soleus muscle, PE values increased above the 0 week control level while the PC values decreased below the 0 week control value. Concomitantly, with these changes in phospholipid distribution were marked increases, at I and 3 weeks following denervation, in incorporation of methyl groups into the methylated derivatives of PE, i.e. PME, P D E and PC. Theoretically at least, this activation of methyltransferase enzymes I and It could explain the alterations in the relative amounts of PC and PE associated with denerration. While this correlation between phospholipid distribution and methylase activation was excellent for the denervated muscles, the findings were less related with the contralateral control muscles at I and

EXTENSOR

DIGITORUM LONGUS M U S C L E

O25 METHYL

o20

TRANSFERASE I

O15 o

left (D) and right (C) muscles. The increased activity of the methyltransferase enzymes subsequent to denervation is in most cases greater in the denervated muscles than in the contralateral controls: however, there are in some cases large increases in methyl incorporation also in the non-denervated contralateral muscles. Although increased incorporation is observed into all of the phospholipids following denerration, incorporation tends to be greater into P M E for methyltransferase enzyme I and greater into P D E and PC for methyltransferase enzyme I1. This was expected since methyltransferase I catalyzes the conversion of PE to P M E whereas transferase II catalyzes the conversion of P M E to P D E and PC. In most cases, the increased incorporation subsequent to denervation was as great or greater in 3 weeks than in 1 week.

v

MUSCLE- D C WEEK-- 0 I P L -P~

3

0

DC I PO~

3

DC 0

DC

DC

PM~

15

o ¢

I uJ

I 12

METHYLTRANSFERASE - - ~ ---

9

'to_

MUSCLE-OC DC DC WEEK- 0 i 3 PLPC

0C DC DC 0 I 3 POE

DE OC DC 0 t 3 PM~

Fig. 4. Methyl group incorporation into phospholipids following denervation of rat entensor digitorum Iongus muscle. See Fig. 3 for further details.

768

PIIII [1, Rosl NBI R(; c! ~d.

3 \reeks tollowing denervation. The methylase enzymes in these right contralateral muscles also showed an actiwttion following denervation of the left muscle (Figs 3. 4; not as great as in the denervated muscle itselfl: however, the distribution of PC and PE in the contralateral muscles m our studies did not significantly change at 1 and 3 weeks follovdng dcncrvation (Figs 1, 2i. Others, however, havc reported changes in phospholipid content and in P(" and PI: distribution in contralateral control muscles as well its in the denervated muscles (Hemer el a[.. 1971: I975: Kabara and Tweedle. 1981). Acti\'ation of tile methylase enzymes and conversion of PE to P M E would be expected to increase membrane fluidity (decrease mcmbranc micrmiscosity)(Hirata and Axelrod. 1978b). lncrcascd membrane tluidity in turn could have drastic effects upon many membranal functions including transport IOverath el a/.. 19701. enzymatic actixitv (Kimelbcrg. 19751. receptor binding (Heron et al.. 1981 ). and intcraction of rcccptors with adenyl cyclasc (Rimon ~,t a/.. 19781. Of course many other factors ~hich v~c h a \ e not exalnined and which may be ahered as a result of denerxation Iplotein and cholesterol to phospholipid ratio: saturated vs UllSaturated fatty acids) can also alter m e m b r a n e fluidit3. It will require further studies to determine whether activation of thc PE methyltransfcrase cnzymes is a primary effect of dcncrx.ation ~\hich in turn leads to other alterations (.,,co Introductionl or whether methylasc acti\ation is itself a secondar\ effect triggered b 3 a more fundamental change associated with denervation. For cxamplc, onc week following denerwnion, the elcctroph>siological differences in the action potentials of fast atld slow twitch tibcrs (extensor digitorum longus and solcus musclesi disappear while with reinnervation these differences reasscrt themselves tMcArdle ~'t el.. 1980). The effects of alteration in melnbrane lluidit 3 upon these electrophysiological parameters should bc studicd m order to detcrmine if these propcrtics are related. It is of interest in this connection that drugs. such as certain local anesthetics, which drasticall> modif,, electrical properties of ncrxc and nluscle membranes also dccrcase the critical transition temperature of membranal phospholipids (Lcs. 1977: Singer. 1977: .Iohnson c't a/., 19791. thereby increasing lluidilv of the membrane. The changes which we have obser,,cd in this paper occur in about the same time frame as man> of the other reported changes follo,aing denervation {scc lntroductionL Following nerve crush, wc obscrvcd transmission loss by the second day. with some recover 3 by the 14th da 3, although functional reco,,crv of

muscle function in the live animal \~,as llol obvious until the third to fourth week. It is also oi interest that acetylcholmesterase acti\itx of tile rat extensor digitorum longus and soleus muscle decreases during denerxation: hov~c~,er, onl\ the scdctlS acetylcholincslcrase overshoots abovc COIltlOI level during reinnerxation {Dcttbarn, 19811. l! will also be noted in our results {Fig. 1 ) t h a t the ",, of PI! also tended to oxcrshoot, abo~e the 0 week conlrol level, a! 5 \~ecks follo~ing dcnervation. Whether these PE changes rcIlect more fundamcntal changes m membrane fluMit 3 x\hich in ltlrn could modil 3 enzymatic a c t i \ i l \ ~ill rcquirc further stud>. Thc actix.itics of the meth\ltransfcrasc enzxmes in the control muscles (0 x~eekl are quite low relati\e to studies on most other lissucs such as. for example. platelcts (Hotchkiss ~,1 al.. 19Sl}. kidnex brush hordcr ( ( ' h a u h a n ~,l al.. 1982) and s.\naptosomcs (Fonlupt c/ a/.. 19821. Thc activit\ of the mcthvltransfcrasc cn/\lllCs is. however, knox~n to varx ~ i d e h bet,aeen tissues: l\~r example brain has onl> about 1",, of the actixit', which is lotlnd in liver IBlusztajn t'l at.. 19791. We thcreli~re had to use optimal conditions in our ass,lt\ s,,stem 111 order lo ha\e stll]]cicnl incorporation of [~H]meth,,1 groups rote ,,he phospholipids. The incubation volume was therefore kept small 10.5 talk the amotlnt of tissue used per incubation was rclaliveh high 150 100 r a g ) a n d the time of incubation long{l h}. It has been shox~n m agreement with our lindings that the actixit5 of mcthyltransferasc is linear l\~r at least I h IHirata cz ,d.. 19781. The entire lipid e x t r a c t [ro111 e a c h i t l c t l b a t i o n w a s a l s o spotted on a single plate in order to h a \ c a maxinmm nunahcr of cotlnls per spot. [ [ndcr these conditions x~e were able to obtain dpm xalucs signilicantly above background even ,filer subtracting for non-specilic incorporation in the presence of 5-AH (the final specilic incorporation m dpm per spot aboxc background ranged 11oi11 a lo~ of 100 in some 0 ~eck control sltmples to a high of about 10.0(10 follox~ing denerxationl. In order to differentiate bet~een methxltransfcrascs 1 and II wc also used conditions optimal for each enzyme since thcir properties differ markedl\ (ttirala and Axchod. 1980f The weight of dcncr\atcd muscle has been rcporled to dccrease approx 50". tx~o weeks following denerx.alion (Hogan cta/.. 1965: Graft {'l al.. 1965a: Hughes and '~ asin. 1'~731 in agreement with our findings of a 68 and 5(Y'. decrease in the soleus and extensor digitorum Iongus musclcs respectively at two weeks liHlowing denervation. Therefore. a gixen weight m the dellel-valed nlusclc represents ~ greater portion of the total muscle than does the s~tnac ~eight in the non-

Methyltransferases in denervated muscle denervated muscle. Nevertheless, the changes which we have observed (Figs 3, 4) are so large that even if the results were expressed as d p m per whole muscle, there would still be a very large increase in methyl group incorporation observed following denervation. The decrease in protein content per mg of muscle was no greater than 20",~ following denervation. Reporting the results per mg of protein would thus not significantly change the pattern noted in Figs 3 and 4. The phospholipid results (Figs 1, 2) were expressed in terms of o~, distribution since this avoided the problems associated with weight changes of the muscle and provided the information required for this study, i.e. whether there is a relative decrease in PE and increase in PC. We do not at this time know the mechanism whereby denervation leads to activation of the methyltransferase enzymes; however, phospholipid methylation appears to be a general mechanism of biological signal transduction (Hirata and Axelrod, 1980, 1982). Alterations in neurotransmitters, trophic factors etc. as a resul~t of loss of neural input might cause the marked increase in phospholipid methylation observed in this study. This perhaps is another form of supersensitivity induced by denervation; that is, the methylase system becomes supersensitive and activated as a result of the loss of neuronal factors which normally regulate this system. Changes in m e m b r a n e lipid fluidity could provide a c o m m o n mechanism for denervation supersensitivity (Heron et al., 1982). REFERENCES Albuquerque, E. X. and Mclsaac, R. J. (1976). Fast and slow mammalian muscles after denervation. Expl Neurol. 26, 183 202. Albuquerque, E. X., Schuh, F. T. and Kaufman, F. C. (1971). Early membrane depolarization of the fast mammalian muscle after denervation, pCliigers Arch. ges. Physiol. 328, 36,50. Albuquerque, E. X. and Thesleff, S. (1968). A comparative study of membrane properties of innervated and chronically denervated fast and slow skeletal muscles of the rat. Acta physiol. Scand. 73, 471 480. Appel, S. H., Andrew, C. G. and Almon, R. R. (1974). Phosphatidyl inositol turnover in muscle membrane following denervation. J. Neurochem. 23, 1077 1080. Artom, C. (1964). Methylation of phosphatidyl monomethylethanolamine in liver preparations. Biochem. biophys. Res. Commun. 15, 201 206. Artom, C. and Lofland, Jr., H. B. (1960). Lecithin formation by methylation of intact phosphatidylethanolamine. Biochem. hiophys. Res. Commun. 3, 244~247. Bartlett. G. R. (1959). Phosphorus assay in column chromatography. J. biol. Chem. 234, 466-468. Blusztajn, J., Zeisel, S. H. and Wurtman, R. J. (1979). Synthesis of lecithin (phosphatidylcholine) from phospha-

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