A chalone in the mammalian lens

E.~p(t, E,qe Itc.~. (1970) 9, G7--72

A C h a l o n e in the M a m m a l i a n

Lens

l I . R e l a t i v e E f f e c t s o f A d r e n a l i n e a n d N o r a d r e n a l i n e on C e l l D i v i s i o n ill the Rabbit I,ens ,%Ht~S);A .I. [A-:~>~ AX[) ,.XIAn~Y J. VOAD~.:~*

Drl,,'tr/,,,',,t ~!/' Pbq.,'ioto!licaL Optics, l.nsOtute of OFhthalmoloq!/, U.niver.sitg of Lomlon, .lmfil N~)'ert, Lo),.,lo,~., IV.C..1, EnftIand ( t?.ec<4o.'d I =1,,lu.,'t 19G9, Lomlon) l:t v i t r o rniltfi ic ~totivily o f ¢ht; rztbl)it, |ens is rt~ve.rsibly i z l h i b i t e d b y ,,ldt't~tudirte~--2 zxg/mt

.~'c)Cit*iVt:l~a|illo i.'4 a p p v o x i n m t~:| 3" (f) (.irt]t:a less efl'e,¢t.ive a n d , izt c e n t fast, t o odJ'Otl,'~lizlo, is ~zo(. ahh~ I,) rost~,)-t~ ( h e a ( : t i v i ( y o f t h e pr(:sUn)l)tivt~ l e n s ebaloJte.

1. Introduction

I'Ixi,i,.~u(.,.. [~.~ ),een obtaiztv,([ for t.hc exist-encc of ~x p r o t e i n in ~he |on.sos o f r~bbir.s a n d ~,~i,;e which, in t.be presence of-~ch'en~fline., r e v e r s i b l y inhibit.s t h e cell d i v i s i o n of ra.bbit, h>tlses i~mib~~ted iu vit.ro (Voa.den, t968; V o a d e n ~ n d Leeson, i970). This Fa'op,..rt,y i~ i~!er~ica.! w i t h t.}uLr, of tl)e el~ido.rm,M c h a l o n e identi_fied b y B u i l o u g h a n d l,~.tu'c)~('~:5 (190-1) a n d sugges~..s tt)a.~ ~Lm itoti(~ congrol n:tecktanism, s i m i l a r t~ t,Imt of ghe skin, i~ii~lxt, exist, also it' the. t~t~Bl~t~u~di,m lens. This is feasibie as b o t h t,issues are d~,,..iv:~tiva.~ of t.}~: .surface e,?.to(lerln. l-{owever, bilat.eru.[ a(Lrenaleetonty of mice., a l t h o u g h leadh~g go a m a r k e d sbimula~ion ,:ff et)ide.rma-I ndtusi.~ (Buil~.mgh ~md l~a,u r e n e e , 1.961. 196G~), has no specific ~.lrt~cL. otx t-.he xnitogio ~mt:ivity of ~he lens for tzp to 3 (l~ys a f t e r tA~e oper~t, ion ( V 0 a d e n an,.' 1.,eeson, 1970). ]nt.et~pretation of t.l)ese da.tr~ m u s t be c a u t i o u s as i t r e m a i n s possibl,:.~. ell.her eh~t, an e,~;Oet, w o u l d b e c o m e ~q?pareng o v e r st l o n g e r t,h n e p e r i o d (~L Teir a m i C~rp6n, 1959) or t h a g o n e or m o r e of t,hc i n t r a o c u l a x tAssues is -. source of a d r e m d i n e . ?d.oreove,', noradrena, line is presenf, in a q u e o u s htiulour, p o s s i b l y bei~Ig ,/erive,l in p a r t .front t.he s y m p a t h e g i c ner~'e p l e x u s of Lhe ciliary Body a n d iris (ef. t)uner, yon Nuler a n d P e r n o w , I954; E a k i n s a n d E a k i n s , 1964) arid. po~entfially ghis h o r m o n e c o u l d r e p l a c e adrex~Miue .iu t h e lens s y s t e m . T h e p r e s e n t oommunicatAon deals wi~;b~ t h e reh~give sensit:iv.lgies o f glm mi~ot.ic cmt.ivity o f ghe r~bbit lens to adz'emfline anti aora(trenaline, i~:. vigro ~nd wi~h t&e abilit.v of noradret~aline t.o n t a i n t a i n t.he i n h i b i g o r y ac~;ivity of ~he p r e s u m p t i v e lens ,:.halor~e. 2. M e t h o d s

S~x--eighl; week old New Zealand ~Vhit,e n~bbits were; used. The action (:~fthe test substances was assessed from ~flie'ireffeeI~ on f~le cell division which .,c.cm-a in l:he epithelium of the rabbit lees asing t.he in vit, ro system, described p reviou.s|y by Frooznberg a.nd Voaden (1966). O)xe h:zss from each rabbit, always served in a contsrol i~mubatAorz. * Prc~_~nI rtdch-ess: l)eparm~e~xt o f P h y s i o l o g y a n d lIiochemi~t;ry, Inst, i t u t e o f O p h t , h M m o l o g y , Llniversii:y of.lxmd,~n, J u d d Ngrcet., Londot~ "W.C. t, J~)agland. 67

68

, S H E I L A J. L E E , S O N A N D MAlt's: ,1. V O A D E N

All l*~n.~es w e r e pr,:-inr-ubate,! tV,r l -2 h r bef~.,re t h e effe:-t- of : t d r e n a i i n e (adren;Lline a c i d burr.rate B . P . ; B . D . I [ . ) o r nc)radrem~li,m ( n o r a d , e n a t i n ( - a c i d tart,'=(te; F r a n k f u r t (MJ--th',e<:hsw'Oer,'nany) wa.~ t e s t e d . T h e i~:~ uhati~,ns we)'e t h e n c,Jntinu,..(t P~r ,,n~: f u r t h e r h,,ur. W h e n t h e lense,~ were m e r e l y "'waMmr.t" w i t h a h o r m o n e t h i s w a s adde,1 t,, the. incul.ati~,~ ~ u e d i u m for 6 nti)t. '['he mixt, u r e was t h e n r e m o v e d w i t l t ~ I'asteu,." pipet:t-.', a.q¢[ fr.,.'~i~ m e d i u m a d d e d in its pla~:r." (.see V(,;,~ten, 19681. B o t h t h e c o n t r M ~,nd l h e te.~t lelm we,..v t r e a t e d s i n t i l a r l v v.xtept t h a t t~,e l)re.~mnptive ¢-hah,n~" w.ts p r e s e n t o n l y in th,. t,esI r,.pla, ,'m<,nt m e d i u , , L In tItese experi'nent.-: t h e i u c u b a t i , m s w e r e c<~,t.inuet~ta'atin:~ri,,,, b y th,. fluori~,tetri,:" m~.~t,h,,d o f Arltfm ~nd S a y r e (|9521. T h i s te~.ll~it{ue tx,ul~[ <,ltlv git'r, a t~-id~" 1,, t h e purit, y. h o w e v e r , as tim Fmseline ,,f t h e a.~-sav w a s p r , , v i d e d b y ;~t,~tb¢'r v,,m:ne:'v-ial prep;trati,2n of t h e h o r m o n e . Nev,.,'thole~s. a f t e r ttv.merm~s t e s t s , t]n. l a t t e r I~',¢t l,~.,.~ acv.epte~[ as adronali~te-fr~e (_-~]~:[)rmale[ :tz,¢l |~aker. [~er~r>~tal ¢-,mt,,mr~im,:i~u~).

t"ra~.l /ol~(~t ;o,t o f gents "p,'otei li.~ T h e p r e s u : r i p t i v e c h a l o l i e . ,e:ts p a r t l y p u r i f i e d fro:v w h o l e t.~xtrat-ts o[' r~,l)t:it~ ft:~l~ b y u-ltilt.ration on S e p h a d e x ().75 u.~itig ~)-05 st '[`ris/HC[ h,~fl'er as t h e ,'.luting .~oIvent (.-.~e %',,adopt, 1968). I t was isolatert b y titis r n e t h o , I wit.h t..he g - c r y s t ~ l l i n s t r u c t u r a l p r o t e i n s rd" th,' len-. "Phe < r u d e fra¢-tion tbtt.a ~ b t a i n : : d ~'at.~ ,li~h':~ed a : - a i n s t at-_.,~.~ ,[ietill,_'~t. w ~ t e r , lyophiliz,.~i a n : l s t o r e d :tt --21)=C. "I'e.st f o r "cA,~la~.e" aeti ,,;t// I t ha3 b e e n s h o w n th~tt a l e n s , a g e d i ~ , vitro{~,r t 2 hr. wil I.nn¢ r e s p o n d t,, t h e il~hil,irorv act, ion ,,f t h e I, r e . ~ u m p t i v e eh;~lone. [ t ' o w e v e r , if t h e lens is the,, was]tvd h r i e t l v wit.h a,1 adrenalilDe s o l u t i m , it wilt, on~:e agai~l, b e c o m e .~ensitive t, t h e i u h i h i t m . v a c t i o n ,,ftb.," p r o t e b t ( V o a d e n , 1968). '['akiug i n t o ,~onsi&ev~tti~,n a l s o t h e effm.ts or th," p , ' o t e i l > on ;, t'reshly iso/atmd lefts, t h e s e t e s t s w e r e used t o asses~ t h e "'(.hai<,ne' ;~ctivits- [-u-,?softt. i[, ~, 7-erystallin preparati,,n.

3. R e s u l t s A dre~.~.diae T i m a < l d i t i o n o f a d , - e n M i r m t o a r a b b i t lens. i n e u l , a t e d in v i t r o , c;mqe,{ ,.n i n h i b i rio,1 o f t i m m i t o t , ic act.ivil)v o f t h a t . len~. T h e til,:<'.-course o[' th.,., i a h i l , i t i o l , ~iv~.:~ I,x" t.h~e q u i v a I e n t o f 2 n g / m l a d r e n a l i n e is s h o w n in F i g I. ~O.~ f - - --~'

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4. Discussiorl

Miki (I96I)),a.-+ e.~t;d)lished t h a t t.t~e m i t o t i c +,LCt.ivlty o f t,hL' )';~t ]en.~ is inhibi(,ed f o l l o w i n g t h e intr~,,vcimus i n j e c t i o n o f 5t) czg adrorm]ine/lOOgms b o d y w e i g l m q'],e l,re:~ent result.,,, a.}t.hol~gh., obtained in vitro. -ix'e_,. sonic: indJcar, ion of t,he .sensitiviLv.. of t h i s r e a c t i o n ......,is l i t t l e as 2 t)g/ml o f a d r e n a l i n e ~:ausi-..ag a 70-8t3% d e p r e s s i o n it~ t.he It.x-el of l~,ito.sis i~ t h e cult, llre(l r a h i ) i t len~ (Vig+ t), A.s 8drenMJr, e is not. b y itself, a ~nitotk: i n h i b i t o r (see B u l l o u g h , 1965. p. 1+388), t~he a b o v e o b s e r v a t i o n s p r o v i d e indii',:ct, exi
:X I ' I I , , % l , ( I X i + :

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15 20 teli~e ;tt't.e'v t h e ;M,lition f (:omit/el,ion o f tnitot.ir tiger('.-,. I,r',-,,-e0l it~ ('i~t" ('I*icl(.rr~is w t w l | t h(: ILor'ttlon~, is .'~,(]~l,4rl, ;l, ric~ ie~c['eus~!s I.bc dm'al:ion only" ¢,l" *b,,<,' u!,i<.l~ <'t,t<-rt,d f~roplms,' ;tfb,r its :~ddit, i,)z~. T h e r e s u l t s a r e ,or. s t r i c t l y c o r . l,;l,'gli.~],'..}1(.~%V1'%~'1". ;.l'4 t!~'I':";i" ;~.ZlI]'}I)}'."; I|NL:I.t fl't'.%lllx" is,~lat(',] t:is.su,~ it, t}mil" e x p e r i m e n t s wl~,,r,,a~, il~ th,' pr,,.se~t, s~ri,'.% :~.t I(,n.~,'..~ w e r e l~re-izwub~te, t ti)r l h r b,:ii)re th,.

. \ ' , , r a d r r t m l i m ' , ¢ , d tDc r , storalio~J ,~./'"cDetl:)tu ,~" acticiQ}

"l'!w ral,Ijit, h,as y-cry.~tallin u~e,I i . ttli.-~ st.u~ly difl'~rs from that, reporLed previously I,:" \',l~l(t~.,I ([.q~iS). ~l~ ;[ r~'~i~hla.l inlti~,it,~rv act.iv!t,: re.,m~i,.~ w h e n t h e In-e-inc:nbat~d all]xl el',' i.~ [e..Iv,~ Ol1 ;1 j,r~.-incuh~Lter~sum pt;ive lens c h a l o n e {'!'able I 1 I). 'l'h(.se result:a, w h i c h ,:-o e.~Se~ltiallv in a g r c , ' ~ e n u w i t h ttw e a r l i e r f i n d i n g s o f B u l i o n g h a n d ILaure~me o n t~,ou:4,: ~,pi,l~.rn~i.-. (.<~e bullou,..h. [962.1 ). 313. 1965, p. ltiStS), i~,l~Iy t,h a t Lhe inhibitor,," a~-t.ion of nora~trez~:tli0~- d o e s n o t a r i s 9 l'ro~l ! h e rv.storaLion o f :t protoi~t h o r t n o n o eo~npl..:x. Thus.

il" a ch;tlo~te-adrenalirm <'Oenlflex l,rovith;s mitot.ie eontrr,1 itt th,' r-tl)bit, b;t~s , . p i t h e l i u ~ l . Lhe. ~tl)ove r e s u l t s M~ow Lh'tl,, it~ tht' l o n g - t e r m aF:serwe Of a ( [ r e n a l i o e : i t o r a d r e l u t l i n e wtmb.[ b~- inefl;.~.ctive as ~ s u b s t i t u t e in t h e sv.<.te~. Althou,,_.,h tb,; p.~',-senl; r,:sull;s h a v e l)ee~ o b t a i n e d wit;h r a b b i t lena p r o t e i n it; is p r o b a b l e thaL t.hey a p p l y g e , e r M / y . A s i m i l a r ~ : c h a l o a e - l i k e " ;tcLivibv has b e e n fom~d ill tno:lse ion s ;.,-cryst~flIin ( V o a d e n a n d ],eeson, J 970)_ ~l oreo v,.,r it },c.~ b e e n est;abJished t h a t LI~o tons a n d e p i d e r m a l p r o t e i n s a c t c o m m u t a t . i v e l y ( L a u r e n c e , u n p u b l i , ~ h e d c, bservation.~; V o u d e n t 9 6 8 ) a n d recent, s t u d i e s h a v e r e v e a l e d t h e w i d e s p r e a d dist.ribut-ion U.lnong v,,.rtebrat.e.n o f c o m p a r a b l e e p i d e r m a l ct~aloae s y s t . e m - ~ - - s b o w i n g l,i.~su,.. r a t h e r t h a n s p e c i e s specifieit.y (Btflldugh. Laui-ence, I v e r ~ e n a n d Nlgjo, 1967 ; ttrondiusBoldingh and l.,anrence, t968). It. d o e s s e e m . t.hervt'ore, that; Lhe }.,rese,tee ,)f n o r a d r e n a l i n e in ~he a q u e o u s h u m o u r ,:anno~ e : : p l a i n w h y b i l a t e r a l a d r e n a l e e t o m y in m i c e h a s no specific effect on l e n s m i t o s i s for u p to 3 d a y s a f t e r t h e o p e r a t i o n (.~ee V o a d e n a n d Leeso~), 1970).

"72

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:'tilLoll, <-\. H. an(~ 5a.y~'e, D. [:'. ([962L .I t'/~(trnmcol. E:cptg Th,:m?. 138, 360. lJullol~gh, IV, S. ([9t;2), Blot. Igor. 37, :](.t7. ltJtl[lougll, \V. S. ([9~5]. C~rm:er Res. 25, t6,'43. tJutlough, W, S. ~t~lc[ l./J,urerico, i!',. I>>. ( 190 ] ). t),'oc.. Ro!t. ,b'(,,:. (L(mdo~) Set. I~ 154, 5.tlt. Bullough, W. S. a i~d l,~rurt~nt:e,, i:':. B. (l!lt;4). E~:ptl (.lell 1~¢:.~.33, 17(i. Bullougl'i, W. ,%. a~xd 1,aurence, E. i l. (196~a). E;cptt (:,:ll Re.s" 43, :{.~:l. .t'>ultough, W. S. tu~d Laure.~lee, [,]. 1". ( ! !)66t)). N~s/~re. 210, 7 t 5. Bnllough, W. S., Laur<;ne~., E. B., [v~r~or~, (). lt. ar, d [':lgj~L K. (] (,}t-:7), .\',L~L,,',~2]4, 57.'4. ])oiler, H., von Eulor, "IT. ~. ~ul(i. I'erno~v, J_,'. (1954), Atwl. l'hffsiol. Sca~uJ. 31, 1 I3. E'aldn~, K, ]i'Z a.nd Ea, kisis, 1-t. M. T. (I9fi4). J..l)harmeteu!. l¢.ep//"l'htmp. 1441, 0t). l;roomberg. D. ~t~l¢[ Voade~l, M. ,]. ilgisf.;), t','xptl E,,,.. l~e~. 5, 1. Flond]us-]Joldingh, W. and I.,v,ureu('e, ~..lJ. (lgti~). E'~ml,. ,/. B/orhe~. 5, t91. Mik.i, '1". (196t2. Actu ,.~;o¢. Oph/h~dmo/. ,l,~p. 65, 22t~7. Teir, H. mid Oa.rpbri, .t. (}.9.59). ,4eta Puthof .tl icrobiot. ,5"r