Ejercicio e inflamacin Increase in blood bradykinin concentration after eccentric weight-training exercise in men CHARLE !

LAI " #R$" % AL!ER& A'A(" % 'E)I (A IC*&&E" + A)' ,RA)C - *I .E / R*))E&0 %,ac1lte / de .harmacie and 0'e /2artement de 3ine /siologie" 4ni5ersite / de (ontre /al" (ontreal H0C 0#67 and +'e /2artement de 3inanthro2ologie" 4ni5ersite / d1 81e /bec a 9 (ontre /al" (ontreal" H0C 0.: Canada !lais" Charles" #r$" Albert Adam" 'enis (assicotte" and ,ranc -ois .e /ronnet$ Increase in blood bradykinin concentration after eccentric weight-training exercise inmen$ #$ A22l$ .hysiol$ :6;0<= %%>6?%+@%" %>>>$A&he 21r2ose of this st1dy was to 5erify the 2ossible a22earance in the blood of bradykinin ;!3< and des-Arg>-bradykinin ;des-Arg>-!3< after eccentric exercise in %0male s1bjects$ Eccentric exercise ;B 0 %@ leg 2resses at %+@C maximal 5ol1ntary concentric contraction< res1lted in m1scle damage and inDammation" as s1ggested by the signiEcant increase in ser1m creatine kinase acti5ity ;from +@F G F% to 0++ G G0 4Hl %+ h 2ostexercise< and by se5ere lasting 2ain" which also 2eaked at %+ h 2ostexercise$ !lood !3 and des-Arg>-!3 concentrations were meas1red by com2etiti5e enIyme imm1noassays 1sing highly s2eciEc 2olyclonal rabbit IgJ$ 'es-Arg>-!3 concentration was not modiEed ;2reexercise= FF G %F 2molHl7 2ooled 2ostexercise= F6 G F 2molHl<$ In contrast" !3 concentration signiEcantly increased immediately after the exercise session ;G: G > 5s$ F+ G 0 2molHl 2reexercise< and ret1rned to basal 5al1es at %+" +F" and F: h ;2ooled 5al1e= F@ G F 2molHl<$ &his obser5ation s1ggests that the inDammatory 2rocess d1e to eccentric exercise-ind1ced m1scle damage co1ld be mediated in 2art by !3$ creatine kinase7 delayed-onset m1scle soreness7 kinins7 inDamation7 des-Arg>-bradykinin EKERCI E" 2artic1larly 1nacc1stomed stren1o1s exercise" eccentric contractions" and contractions 2erformed at long m1scle length can res1lt in delayed-onset m1scle soreness ;'*( < ;for re5iew see" e$g$" Refs$ %" 6" %F" %6" +%" +F" 00" and 06<$ !rendstr12 Lsee Armstrong ;%<M was a22arently the Erst a1thor to s1ggest that '*( co1ld be associated with an inDammatory 2rocess triggered by exercise-ind1ced m1scle damage$ 'amage to the m1scle after 5ario1s ty2es of exercise has" indeed" been demonstrated fromdirectmor2hological e5idence ;%G" %6< as well as from changes in a

n1mber of markers of tiss1e damage and re2air$ er1m creatine kinase ;C3< concentration andHor acti5ity are the most common markers 1sed ;e$g$" see Refs$ F?G" :" %:?+@" +B" +:" +>" 0G" and 06<$ E5idence of cell disr12tion and of increase in collagen and myosin t1rno5er has also been shown by 1sing other ser1m or 1rinary markers" e$g$" as2artate ;B" +>< and alanine aminotransferases ;B<" alkaline 2hos2hatase ;F" B<" lactate dehydrogenase ;F" B" +><" carbonic anhydrase ;0><"myoglobin ;:" %>" 0><" myosin hea5y chain ;+B<" galactosylhydroxylysyl gl1cosyltransferase ;0><" hydroxylysine and hydroxy2roline ;F<" and 2re-2ro-collagens ;0><$ &he hy2othesis of an inDammatory 2rocess in the m1scle as a conseN1ence of this damage is consistent with the major signs and sym2toms obser5ed" s1ch as m1scle 2ain ;i$e$" '*( <" swelling" and the loss of f1nctions" s1ch as red1ction in Dexibility and strength ;6" +F" 06< b1t also red1ction in ins1lin sensiti5ity ;%><" the ability to store glycogen" and end1rance ;+<$ 'ata concerning s2eciEc histological and biochemical markers of inDammation are also in line with this hy2othesis= changes in 2lasma white blood cell s1b2o21lations ;B" G" 0>< and inEltration of white blood cells in the m1scle ;%G" %:" +0<7 increase in 2lasma ;B" %:< and tiss1e cytokine concentrations ;%G<7 and acti5ation of the com2lement 2athway ;G<$ &he recent re2ort ;%:< of an acti5ation of xanthine oxidase in the micro5asc1lar endothelial cells of m1scles and in inEltrating le1kocytes after exercise is also consistent with the hy2othesis of an inDammatory 2rocess$ ,inally" this hy2othesis is also consistent with the obser5ation that anti-inDammatory dr1gs co1ld red1ce '*( and some of its associated signs ;see" e$g$" Ref$ ++<$ e5eral models ha5e been 2ro2osed to ex2lain how exercise co1ld trigger an inDammatory res2onse in the m1scle ;%" %F" +F" 00" 06<$ A common feat1re of these models is the in5ol5ment of 5ario1smediators of inDammation" incl1ding histamine" serotonin" nitric oxide" cytokines" 2rostaglandins" oxygen free radicals" and kinins$ Howe5er" exce2t for some cytokines ;B" %G" %:<" the in5ol5ement of these 5ario1s 21tati5e factors in the res2onse to exercise-ind1ced m1scle damage and in '*( remains hy2othetical$ &he 21r2ose of the 2resent ex2eriment was to 5erify the 2ossible a22earance of bradykinin ;!3< and of its acti5emetabolite des-Arg>-bradykinin ;des-Arg>-!3< in the blood after m1scle damage" triggered by eccentric exercise and associated with '*( $ !3 and desArg>-!3 are 2owerf1l mediators of inDammation ;%0< and" as s1ggested by Armstrong ;%< and by (acIntyre

et al$ ;+F<" co1ld be in5ol5ed in the 2rocesses linking exercise-ind1ced m1scle damage to inDammation and '*( $ (E&H*' 1bjects$ &he ex2eriment was cond1cted on %0 acti5e and healthy male s1bjects who ga5e their informed written consent to 2artici2ate in the st1dy" which was a22ro5ed by the Instit1tional !oard on the 4se of H1man 1bjects in Research$ &heir age" height" and weight were ++$6 G @$G ; E< yr" %66$+ G %$6 cm" and 6B$0 G %$: kg" res2ecti5ely$ )one of the s1bjects was taking 2art in any reg1lar training 2rogram at the time of ex2eriment$ &he costs of 21blication of this article were defrayed in 2art by the 2ayment of 2age charges$ &he article m1st therefore be hereby marked OOad5ertisementPP in accordance with %: 4$ $C$ ection %60F solely to indicate this fact$ Exercise$ &he s1bjects 2erformed a weight-training exercise session on a s12ine leg-2ress training a22arat1s ;&rotter" (adway" (A<$ &he exercise consisted of E5e sets of %@ eccentric contractions of the extensor m1scles of the lower limbs at %+@C of the maximal strength meas1red in concentric contraction" or %$6G G @$@6 k)$ ,or this 21r2ose" the maximal strength" which co1ld be de5elo2ed d1ring a single concentric contraction" was meas1red in a 2reliminary test session % wk before the ex2eriment ;%$F6 G @$@G k)<$ &he day of the ex2eriment" two aides lifted the weight in the 122er 2osition on the leg-2ress a22arat1s while the s1bject extended his lower limbs against no resistance$ &he s1bject then lowered the charge as slowly as 2ossible$ Each eccentric contraction took "0 s$ A B-s rest 2eriod was allowed between the contractions while the weight was lifted again by the aides to starting 2osition$ A B-min rest 2eriod was allowed between two consec1ti5e sets of contractions$ &he entire exercise session" which incl1ded %@ 1naided concentriceccentric contractions at B@C of the maximal concentric strength as a warm-12" lasted "FB min$ (eas1rements$ &he day of the ex2eriment" obser5ations and blood sam2ling from an antec1bital 5ein ;%@ ml< were 2erformed at rest before the beginning of the exercise session ;between G=@@ and 6=@@ .(< and immediately after the exercise 2eriod$ *bser5ations and blood sam2lings were also taken %+" +F" and F: h after the end of the exercise session$ ,or the meas1rement of C3 acti5ity" B-ml blood sam2les were centrif1ged ;+"@@@ g" %@ min" FQC<" and the ser1m was stored at ++@QC 1ntil analysis$ er1m C3 acti5ity was assayed by 1sing an a1tomatic enIymatic method ;Hitachi 6%6< with commercially a5ailable reagents ;!oehringer(annheim<$ ,or the meas1rement of !3 and des-Arg>-!3 concentra-

tions" B-ml blood sam2les were immediately collected in 2oly2ro2ylene t1bes containing %B ml of ice-cold ethanol$ &he t1bes were shaken 5igoro1sly and left to stand in cr1shed ice before centrif1gation ;+"@@@ g" %B min" FQC<$ &he clear s12ernatant was collected and e5a2orated to dryness ; 2eed Rac Concentrator" a5ant" ,armingdale" )S<$ &he resid1es obtained were res1s2ended in + ml of triD1oroacetic acid ;@$%C 5olH5ol< and 21riEed on a %-ml Taters e2-.ak Rac C:-silica cartridge ;(illi2ore" (ilford" (A<$ &he el1ates were then e5a2orated to dryness" and the resid1es were stored at +:@QC 1ntil analysis$ Imm1noreacti5e !3 was N1antiEed in the resid1e res1s2ended in F@@ Ul of a &risVHCl b1ffer ;B@ m(" 2H 6$F<" containing )aCl ;%@@ m(< and &ween-+@ ;@$B mlHl<" by a com2etiti5e enIyme imm1noassay ;%@<$ &his assay 1ses highly s2eciEc 2olyclonal rabbit IgJs raised against the carboxy terminal end of !3" digoxigenin-labeled !3 as tracer" and alkaline 2hos2hatase-labeled ,ab fragments antidigoxigenin" with the s1bstrat 2-nitro2henyl 2hos2hate to detect and N1antify the imm1ne com2lexes$ &y2ical calibration c1r5es for !3 were characteriIed by a half-maximal sat1ration 5al1e of @$6: 2molHml ;%@<$ Imm1noreacti5e desArg>-!3 was N1antiEed by 1sing a similar analytical a22roach to that described by Raymond et al$ ;0F<$ (1scle soreness was assessed for the right N1adrice2s m1scle on a fo1r-le5el scale= @ B no 2ain7 % B 2ain when 2al2ated7 + B 2ain when contracted7 and 0 B 2ainf1l at rest$ tatistics$ Ral1es are means G E$ Com2arisons were made by 1sing analysis of 5ariance for re2eated meas1res and a &1keyPs 2ost hoc test$ tatistical signiEcance was set at . " @$@B$ RE 4L& er1m C3 acti5ity and !3 and des-Arg>-!3 concentrations in the blood before and after the exercise session are shown in ,igs$ % and +" res2ecti5ely$ Immediately after the exercise session" ser1m C3 acti5ity was slighltly ;%> G 0C< b1t signiEcantly increased abo5e basal 5al1e$ &he 2eak 5al1e was obser5ed %+ h after the exercise session ;GF G %BC" with wide interindi5id1al 5ariations= range ++@ to %%6@C<$ er1m C3 acti5ity decreased thereafter and was back to basal 5al1es F: h after the exercise bo1t$ !lood !3 concentration a5eraged F+ G 0 2molHl before the exercise session$ A signiEcant G: G +6C increase was obser5ed immediately after the exercise session" alsowithwide interindi5id1al 5ariations ;range= +F6 to %00@C<$ !lood !3 concentration was back to basal 5al1es %+ h after the exercise session and remained 1nchanged thereafter$ &he 2resence of des-Arg>-!3 was detectable in the

blood in only 0: of the GB sam2les analyIed ;B:C<$ )o signiEcant change from 2reexercise 5al1e ;FF G %B 2molHl< was obser5ed immediately after exercise or on the following days ;2ooled a5erage 2ostexercise 5al1e= F@ G B 2molHl7 n B F+ sam2les<$ e5ere 2ain de5elo2ed in the N1adrice2s m1scle immediately after the exercise session ;,ig$ %< and f1rther increased to reach a 2eak %+ h after the exercise session$ ome of the s1bjects were still ex2eriencing some 2ain 6 days after the exercise session" when a follow-12 informal s1r5ey was made$ 'I C4 I*) e5ere and lasting 2ain in the extensor m1scles of the lower limb was ind1ced by the high-intensity eccentric weight-training exercise 2erformed$ &he 2ain" which was 2resent immediately after the exercise session b1t increased the following days" with a 2eak at %+ h after exercise" was ty2ical of '*( ;F" 6" :" %:" +F" +B" +:" +>" 0G<$ Associated with m1scle soreness was an increase in ser1m C3 acti5ity" which was a22arent at the end of the exercise session b1t was more marked %0 and +F h later$ Increase in ser1m C3 acti5ity or concentration is a 5ery consistent Ending associated with '*( ;e$g$" see Refs$ F?G" :" %:?+@" +B" +:" +>" 0G" and 06< and is generally considered amarker of exerciseind1ced m1scle damage ;+@<$ &he c1rrent hy2othesis for ex2laining '*( "m1scle swelling" and loss of m1scle f1nctions after exercise s1ggests that exercise-ind1ced m1scle damage triggers an inDammatory 2rocess ;%" +F" 00" 06<$ Altho1gh this hy2othesis is consistent with most of ex2erimental e5idence ;see the introd1ctory section<" direct e5idence of the a22earance of the classic mediators of inDammation remains limited" exce2t for some cytokines ;B" %G" %:<$ Endo et al$ ;%B< ha5e recently re2orted in mice that exercise increases the acti5ity of histidine decarboxylase in the m1scle" s1ggesting that histamine co1ld be in5ol5ed in the de5elo2ment of fatig1e and '*( $ In f y " e t " n t 0 r

d " e e s s l e " t e n contrast" in5ol5ement of 2rostaglandins ;:< and oxygen reacti5e s2ecies ;:" %:" 0G< has 2ro5en diffic1lt to ascertain$ &he 2resent st1dy was 2erformed to 5erify the 2ossible a22earance of !3 and des-Arg>-!3 in the blood in res2onse to eccentric exercise res1lting in m1scle damage and '*( $ !3 is formed from kininogen 2rec1rsors 12on the action of tiss1e and 2lasma kininogenases" which are acti5ated by 5ario1s stim1li" incl1ding tiss1e damage and inj1ry" anoxia" and acidosis" whereas its acti5e metabolite des-Arg>-!3 is 2rod1ced 12on the action of kininase I ;see Ref$ %0 for a re5iew<$ !3 and des-Arg>-!3" or kinins" acting on !+ and !% kinin rece2tors ;+6<" are 2owerf1l 2roinDammatory factors$ &hey 2rod1ce local edema and acti5ate and sensitiIe sensory and sym2athetic ner5e endings" with the release of other mediators of inDammation ;e$g$" 2rostanoids" s1bstance ." ne1rokinin A" and calcitonin generelated 2e2tide<" which mediate some of the local action of kinins and am2lify their 2roinDammatory" nocice2ti5e" and algesic effects$ 3inins also attract le1kocytes" acti5ate the 2hagocytic f1nction" and increase 2rod1ction and release of inDammatorymediators fromne1tro2hils and macro2hages$ In ex2erimental models of inDammation in rat" !3 2lays a major role in the de5elo2ment of the ac1te inDammatory 2rocess ;><" whereas both !3 and desArg>-!3 are in5ol5ed in the de5elo2ment of s1stained inDammation ;0<$ !3 co1ld also be generated in the exercising m1scle ;%+" 0%" 0B" 0:<$ Te th1s hy2othesiIed that !3 co1ld 2lay a role in the ac1te de5elo2ment of m1scle inDammation after exercise-ind1ced m1scle damage" whereas both !3 and des-Arg>-!3 co1ld 2lay a role in the s1stained inDammation obser5ed the following days$ Te also hy2othesiIed that

the in5ol5ement of these 2e2tides in the inDammatory 2rocess in the tiss1e co1ld be reDected by changes in their concentration in the blood$ !3 concentrations in blood sam2led before the exercise session ;F+ G 0 2molHl< and at %+" +F" and F: h after the exercise session ;2ooled a5erage 5al1e F6 G F 2molHl7 n B F+ sam2les< were slightly higher b1t consistent with basal 5al1es obser5ed by .ellacani et al$ ;0+<" who 1sed 5ario1s methods of extraction and assays ;%6?0G 2moHl<" and in o1r own laboratory ;+>G> 2molHl< ;+G<$ In hemodialyIed 2atients" des2ite the inDammatory 2rocess triggered by the dialysis membrane" !3 concentration in 2eri2heral blood was not signiEcantly modiEed +@ min after the start of dialysis ;+G<$ In contrast")1ssberger et al$ ;0@< obser5edmoderate ;0+< -to-large increases ;00B< in blood !3concentration in 2atients with angioedema$ In the 2resent ex2eriment" immediately after the exercise session" !3 concentration in 2eri2heral blood also increased abo5e basal 5al1es obser5ed before exercise or %+" +F" and F: h after the end of exercise$ &he a5erage res2onse remained modest ;GF G %BC<" and wide interindi5id1al 5ariations were obser5ed ;range of change= from +>to >: 2molHl<" b1t the increase was statistically signiEcant and was obser5ed in %@ of the %0 s1bjects$ &his transient increase in blood !3 concentration immediately after exercise is a f1rther s122ort to the hy2othesis that eccentric exercise triggers an ac1te inDammatory 2rocess that co1ld be mediated" at least in 2art" by the generation of !3 in the exercising m1scles with a s2illo5er in the blood$ In contrast to the transient change in blood !3 concentration" no signiEcant change frombasal 2reexercise 5al1e was obser5ed for des-Arg>-!3 immediately after exercise$ &his obser5ation is consistent with the res1lts from o1r laboratory" s1ggesting that the con5ersion of !3 into des-Arg>-!3 in the blood is only a minor 2athway in h1mans" contrib1ting only "0$BC to !3 degradation ;%%<$ !lood !3 concentration had ret1rned to the basal 5al1e %+ h after the exercise session and remained 1nchanged thereafter" and no signiEcant changes in des-Arg>-!3 concentration were obser5ed$ &his co1ld indicate that none of the kinins are in5ol5ed in the s1stained inDammatory 2rocess after eccentric exercise$ An alternate and more likely hy2othesis is that kinins are in5ol5ed in this 2rocess b1t that their generation in the inDammed m1scle does not translate into changes in their concentration in 2eri2heral blood$ &he 2resent st1dy does not allow 1s to ascertain the

mechanisms by which the eccentric weight-training exercise 2erformed ac1tely increased !3 concentration in the blood" the so1rce of !3 fo1nd in the blood in higher amo1nts after than before exercise" and the 2ossible role of !3 in the de5elo2ment and maintenance of m1scle inDammation and '*( $ Isometric m1scle contraction for 0@ s" which is 1nlikely to res1lt in m1scle damage" is associated with the generation of !3 in the tiss1e ;0:<$ &his s1ggests that the a22earance of !3 in the m1scle co1ld be a normal e5ent" b1t the mechanism by which m1scle contraction locally triggers the kininogenase-kinin system remains 1nknown$ &his mechanism co1ld be res2onsible for an increase in tiss1e !3 concentration with a s2illo5er in the blood in res2onse to the high-intensity eccentric exercise 2erformed in the 2resent st1dy$ Additionally" tiss1e kininogenases co1ld ha5e been acti5ated by s1bstances released from disr12ted cells or extracell1lar str1ct1res as a conseN1ence of exercise-ind1ced m1scle damage$ &he res1lting b1ild12 of !3 in the tiss1e co1ld ex2lain that it esca2ed inacti5ation and leaked into the blood$ 4nder this hy2othesis" the increase in !3 concentration in the tiss1e co1ld 2lay a role in the de5elo2ment of an inDammatory res2onse" incl1ding 2ain$ Alternately" !3 co1ld ha5e been generated in organs and tiss1es other than the exercising m1scles" from which it co1ld ha5e been released$ ,inally" it cannot be excl1ded that !3 was also generated in the blood as a conseN1ence of acti5ation of 2lasma kininogenases by 1nidentiEed s1bstances released from the m1scle andHor other tiss1es$