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ATPase activities in retinal pigment epithelium and choroid
Exp. Eye Res. (1978) 26, 445-455
ATPase Activities in Retinal Pigment Epithelium and Choroid
The Ilg”--. the Sal--K&and the HCO;-dependent i\TPase activities of retinal pigment epithelinln~choroid tissue from grassfrog, bullfrog and rabbit, have been measured. In the frogs, X’Ig”?~ ATPase activity was het,neen 1 and 2 ~~mols Pi liberated hr-l rng--l protein, while Sn-l--K+ ATPase and HCO, ATPase were about PO and %“,, of this activit,p. respectively. Corresponding values for the mbbit were X.5 pm01 Pi hr--’ mgpl prot’ein. HO,, and WC,. Sal ~.K+ ATPast, activity in each species \vas completely inhibited b>l 2 mll-calcium. ~vhilr H(K1, ATPase was llnaffected by calcium or hy ouahain. The pigment epithelium-choroid preparations of bollfrog and rabbit were scraped to yield suspensions of isolated epithelial cells. relatively free of other t,isstle component,s: and and red blood a residual fraction which contain4 some’ rpit helial cells. c~horoidal elements ~4ls. >Ya- -Ii+ ATPasa activity was present in t,he epithelial culls of both species and absent from the choroid of the rabbit. H(‘OJ XTPnse and IrIg’ 1 ;\TE’nse activities were ronghlg bl(t in thr rahhit xverr concentrated in the equally dist,rihllted in the hnllfro, u fractions. choroid. The result,s are discussed in relation to the postulated active ion transport pr~xcsse~ of the pigment epithelium. h-p!/ forces: pigment epithelium ; choroid; ac+ivc trallxport : >I$‘~ .ZTPase; SR’ -I< .\Tl’asc; HCO, AITPasr; orlabain : cal(Gllm.
1. Introduction The pignlent, cpitheliuni performs a nninher of functions which xe essential for the survivd of the photoreceptor cell and for the rneint,~?na.nce of retinal responsivrwess to light.. TINS. the pigment epit,heliultl hell’s to control t,he lengtll and shap of the outer segment of’ the photoreceptor cell 1)~ R conq~les process of phagocytosis of the tliStil.1 til)s of thcl outer segment discs (Young itilt JZol<, 1969) and it regubtes the flow of inolecnles and ions hetwwn t,lie choroidal hlooti ant1 t’he neural retim (Socll. 1952. 1963; Lassnsky and DcPisoh. 1966; Steinlwrg ant1 Miller. 1973: Niller and Steinlwrg. 19i6. 19’iia.l)). IOIl fnscs across isolated pjgnmit cpit’heliunl -choroit’l ~)rqssmtiutis have heen nieasu r-et1 in the itlweuce of an elect~rrxhenlic;%l ]wtcIltial t~ifftwnce in order ix delmxiine which ions are actively ~r~tllSJ)~Jrtcd by the pignicnt~ tyit~tielilln~. A net transfer of chloricle froni t’he retina to t’lie choroitl ztnd a net t.miisfer of sodiuu1 from the choroid to the retina \i-ere found in the tmd (B’clfo ~r~~~irrus) :mtl ltullfroy (RUM c:trtesbeinm) prqmmtions (l~;tsansl<>~ ant1 I>eFisch. 1966; Miller and Stein1~er.g. 197’i;i.). In hot11 thrw slwcies the twm short circuit, current could not Iw ent,ircI!s
?Lccouutecl
for
fiv
the
nrht. fluxes
of srrdiurll
~(1
chlorj(le
a.ntl
it. WAS suggest,4
that, at1 active H(‘O;/H-+ transport s,vstenl Inay contAMe bo this current. In contrast, to tlicsih studies 011 colrl~t&~otletl verte\)mtes. Noel1 (1963) xntl Nnell. C’rqq)er ant1 Paparlc~lli (1965) found evidence of a,n active transport of potassium from bhc choroitl t,c)\varti thc& vitreous huxnor which a,ccowt,etl for a major fraction of the short circnitv curvent across the Ijignlent epitheliunl in the ral)l)it. That ount)ain, when added to t.he apical (rebinal) side of the isolated pigment, epit’heliulu-choroicl preparation of t,he frog. a,lters the net flux of sodiunl across the pigment, epit’helium (Miller and Steinberg.
2. Materials
and Methods
C+rassfrogs (Rtow f)iljie~ln) a1tc1 bullfrogs (I/(ol(( wfwhf~i~~)~~6) were 11;t1,1; ;t(laptecl for sc\-em1 hours and decapitated. The eyes were removetl, bisected equatoriall~mid the> WJII hiued retina-choroitl n-as e;~rcfull~- rlissect,etJ away frort~ the scleru. The IEUIX~ retimr GIS t,hett gently clet.;whetl from t,he remaining JGgment epitheliulll choroicl tissue. This procetlure is similar to that nsecl bv T,aruttskv nntl DeFisch (1966) ant1 Miller ;r~ul Steittbrr,g (19771)) for obtaitiitlg the pigmertt epitheliu;Il choroitl free from other ocular tissues. I’ignletttetl rabbits were killecl by ;tir etnbolistll i~ttd the eyes \\‘ere ettwleatetl. Tlrra posterior part of the eye was freed c~f vitreous humor x~ittcl cut iJJt,o li;llves through the optic tterre. The neural retitiit tloate(l front t,lie retrraitiitlg tissue after :L short time it1 1tor11ts1 buffered saline. The pigtncrlt epitl~elint~l~c~l~or(~i~l wits then separ;itetl t’ro~r~ t,hr ac,Jtw by severiug the blood veswJs \vitlt FiskeforceJ)s ~ritd liftiiig ofi the isolated J”.V~~ilJ’~l~iOll. Tlte iigment epitheliunt -choroid frottt cac11 of the species was hotnogertizetl it1 water, usitig it, glaw pestle atid t,ul,c. W;r.trr \ws choseit its the Iiunrc~gei~iziiip medium tlcspite t,he ,liSilllV;lllt;t~e of aggreg;itioJt of cell c~~~rirpot~eiit:Y sometitiiw observed with this ~~rowclure (\\ Iiic,lJ triight, il~COUllt for the r;iriiition iii &T-it! htrl ottp pJ~epitr~tti011 to ;ttlc>tlleJ~). 1‘llt~ a~clv;iJititge Of ~~~ilte~ \IibS tllilt the OsttiOt~i( shock lvsetl cells ;ttirl clisperswi JGgnwtlt gr;liiules. givinfi it higher specific, activity th;ttl Jlotlto,~ett;tt,es in \~ufkr. without twcwt 10 .~h~~ltll)ilia;ttio~~” wit,11 detergents. Tissue front tn.0 ,grwsfrog or bullfrog evt~s \V;LS uwl for 2 1111 of hotnogetiate yielding itui it\-rra,ge protein couc~ettt,r;ttic~r~ of 0.4.; ttig: ‘Itrtl for* gtxssfro,g ;r11t1l.tt ntp/~ul for billfrog. For t,lre r;rhhit, tissue frollt otle half of:tlJ eve \V;LS hOlll~J~etLiZ~~~~ jli .I ml of wter , giving itn :L\‘erxge protein cottc~rtttr;itiotl of 0.41 tltgitttl. .\ crtttlc sep;rr;~tiott of the IJigt1Jeitt rpitdteliuttk -cltoroi~l ittto its c~c)llJlmtleltt ]kart.q \vils ;~lso ;~Ctemptetl. After t,lic wtltbinetl prcJxtr:itioti w i 1+ isol;rtrtl frotll the s(‘Ier:L at)11 treUJ’;l~l rctili;t it \YitS phcetl witli ;I, fe\v (lrops of J~hvsiol~~~ic;~J wlinc 011 i, fht surface untlct~ :I dissec,t,itig tnicrosaope. L\ sp;l,ttil;t \viiq use11 to push c,luttrJw of pigtnctttj epit~lielial (*ells frotii t,hca ttt~clerlyiug choroid. C’ells cattle free itt sheets. stltitll grouJx6 ittt(1 seJxtx;ltely, but f’e\\free l)iptetlt grandes wew ohrn~etl. ittclic,;ttittg th;tt few cells \vcre brl~keii /)I’ this J)t’Ouetliiw. :In alternativr tnethotl. hlretlt,I\r sh;tkill,c tlte tissue itt it st11;11l t,ot.tlr (.otttainitt~ s;ilitict itiicl several KlitSS I,e;r,ds, resultcat! iii fair rrlllov~ll of cells front t)llr choroid;tl tissue but excessive dispersion of pigitietit gr;itmles, supgestitrg that contents I,oth of epithelinl c~,lls :1,t1(1c.ells from the choroitl were included iti the suspension. After scrapittg, t IlC isol;ttwL c.ells were removetl with t)lie c)verl,viilg flui, ;~lttl hnniogettizetl :11ttJtlilutc>cJ :tppronxs also hotrtogenize~l itt s;rlitlcl. pri;ttely. The retnailtillg t,issuc, clesigtlatetl “choroi~l”, III tlie caqe of the bullfrog, this tissue wit,s still hearily piglrtetltetl, p:irtlv l)>- rcittaittittg epiBJielia1 cells (estiiiiat,ed 3s 20’: I1 of total) iLll(l bv t,he pigtuellt stirroutitlitig tile bl0d ve,qsels (see Fig. 1). The rabbit rhuroid. having 110 pigmetlt a11(1 lvitq Hitt on tltc’ scJrr;t. ~~nltl 11e scrap&l virtually free of epitlielial cells.
.ITPxse activity was measured by- determiuing the phosphate according to the method of Fiske and Subbarow (1925), as modified
liberated from ATP bv Winkler and Riley
ATI’ASES
IS
PIGRIEKT
EPITHELIl’~l
hS1)
(‘HOROID
117
(1977). The incubation medium contained Tris-HCl, 50 111~; NaC!l, 120 mx; KC& 5 111~; ATP (Sigma Chemical Co., St. Louis, MO.) 2 nm: MgSO,, 2 rnx; awl EDTA, 0.3 HIM. OuabitiIr. when present, was 0.1 nm. Sodium bicarbonate (‘25 nm) was substituted for all eyuivnlent amount of NaCl when HCO; ATPase was determined. The term HC’O; hTP;w is used itlstead of aniorl-stusitive ATPnse bemuse bicmbona.te is the only IN:~~IU ;wion ~lther thm chloride fouud in intro- amI extrircellular fluids. However, t,he enzyrlw activity of the pigment epitheliun-choroid preparation was also stimulated by wltit~~ ;t~r(l zl11~et1 minor variations when other anions or sucrose were substituted for chloritk. pH \v:I.~ brought to 7.7 for frog esperiulents and 7.4 for rabbit experimeutn. Althcqgll 2.5 n111 NaHCO, is not, compatible with these pH values without bubbling with an appropr.iate tmrtial pwssure of CO?, it was observed that such bubbling did not change thl* a(,ti\-it\ of the preparations. It was concluded that significant loss of C’O, from the nlelliuul tlill Ilclt Iwwr during the period of illcubation. .\lic~not.x of homogenate of 0.1 1111were used in final irssny volumes of 0.5 ml. triplic-xtt. srta ol’t ulws being used for ewh set of conditions for a given homogenate. Frog prepar;Lticlw ~ve~‘t~ illcxhhetl at 22°C for 40 min. rxhbit preparatiolls at 37°C for 30 min. The extent ot ATI’ It!xlrolyis was less than iLO’),, and was linear with time below this value. It \Y;I~ foulIll ttmt the specific activity of a given preparatiou declined by ~8I’:(, over a fourfc&l r:r~il~:l~ 01’ proteiri c~oiicentratiou in the assay medium, but’ wit’hiri ;I single species the v;lri:lticlti of protein in honiopenates wiis ilever greater th;lii t\rcbfr,ld.
3. Results
VII* ;:lqmtra~~~ of the pigment el’itheliunl-chor(li(l lml~aration cbl)titiuetl f’rollr the I)ulli’ro~ is show) in Fig. 1. The epithelial cells. most hearilp pigmented towmls t,llca apica, Glcl. lie in a single uniform layer across t’he yltrfacc of the tissue. support,wl II\T vasculosum, heavil! l)ig~~~t~t!tc(l and filled with red l)lood cells. In some se&ions apical villi were clearl\ sw11. ! rut tmhcse appeared to collapse and flatten to the surface of the el~ithrlial ct>li> ilr ~r~cl
‘l’tl(l isolated epithelial cells of the hullfrog are shown in Fig. 2. It, alq)ears t,llil,t ;I. sing11~ cell Iaver is present, the cellsprobahl\being held together 1)~ Bruch‘s mc~nl)r;~m~. Ilnt it. is tlclt certain whether parts of tie choriocapillaris arc ‘Ml attachctl ~IXIIII Fig. 1 this wonld appear to be the most likely site of cleavage. The free cells are clra,~l! clev~ li(l of ot#her tissue components. Few red I)lootl wlls antI few free pignwntj gr;~lnll<~~ ww
a ~qmreiit~
in these
suspensions.
nit> specific mtivities. based ou protein collceiltri-ltion. of the &I$ -- ant1 iTi1 1-K st,illllll;ltetl ATl’ases of the pigment epithrrli7llll~c.floroi~l frank t,he three espcri~lwntai mill& we sh~~wn in Table I. The colcl-~~lwcled auinmls showtl low R4g’j ATPaw activitiw and when ouahain was omitted from the awav medium there was a sltlall iin(I +nificant increase in the rate of ATP h+)l+s ‘indicat’ing the presence of a the activit’y of t)his enz,vme was about lV)b of t’ht, Ka I< ATPase. In the bullfrog. Mg’: .ZTPase xctivitv and in the grassfrog about 200,,. The specific activities of t,lw &tg”! ;7nrl h’a-’ -K+ ATPases in the pigmmtetl ralbbit w,re alqmsinmtel~~ six- amI
variability from experiment to exl~eriment in the Mg 2+ ATPase activity, the ortGsion of ouahain always led to an increase in t,he rate of ATP hydrolysis. This increase in ATPase activity (Na+-K b ATPase in Table II) was shown to he significant using the method of t#esting the significance of the mean of differences betwcen the Mg2+ and iMgz+ +Na+-Kf ATPase values for each experiment; this procedure was justified
ATPASES
Frc after cells.
IN
PIC:.\IENT
EPITHELIIJM
AND
CHOKOID
4.ilI
11. \‘.
1:ILk;s
lq:‘I‘ .\I.
since each pair of values was obtained front. a single hon~ogenattt. This tnethotl is particularly useful for denlonstrat,ing the significance of small tliffcrencw whrtt variability between individual preparations is of a magnitude cotnparal~lo to t,lte crl)servcd changes in AT&se activky when mat)ain is omitted. This is I twst. iLl)l)a~rt~I~t in the case of the Imllfrog when a sinlple conq)arison of the differelm of the nLeau5 leads to a P value hy Stujent’s t-test’ of appxiinately 0.1. T1irrrfi.m. we have: t~sctl t,lic mean of t)lie tlifferenccs in t,he statistical atmlyses throughout. Table IIT. giving results of c~xprinrents in the presence of 2 tt~hi calcirm. Aows that8 this ion c~Jlll#!te~y inhihitr; the Na~,--K ATPase activity of’ the Ijnllfrog l)rt’l)arat,iun. Again. i\Ig”-~ ATIke a,ct,ivity varietl twtwen homogcnatc-. Imt in no caw
“43lI 1.61 14s I.05 14;i
II.91
(I..34 Il.76 (I.,? I I I.!%
-‘.S4 I ,iT 2.1-i 1.21’ I.filI 1.tl.i I kill II.!)-1 Il.;? I.12
452
)I.
\‘.
RILEY
K’J‘ AI.
Homogenates of tSltr cottthincci pigttteub c]tit,he]iutn--cltoroill anti of t tttt sca]“tt’it tcstl fractions (SW below) all cotttainetl 4pnificattt atttount,s of l11011tl.To etitttittatv t 111, ])ossibilitv that this co~q~onerit of t,llc hOttiOgwat~cs cOith’i~lllt~?~~ sltil~tiLtitid1\’ 10 ittl\ of t)he nteasured ATPase activities. qu\])(Gotts of’ frog an(i ritl~l)it I~loc~tl.(‘ont:linitl< rttuclt higher cottccnt~rations of reel I)lootl cells t’hatt in the t,issut, ]~~t~l~;t.t~atic~t~~. \\(‘I’(’ also assayed. The l\;lgz* ATPast! act#ivity of frog t)too(i \\-its 0.15 ptt101 I’i’ltr/tlt,ti probein (average of five experiments). anti of rabbit8 l~looci was 04 ~ntol/ttr;‘nrg protein (average of four experiments). an(l the Xa- K - and H(Y); .\‘I’Pnsr a(+tivitif,q were negligible under the conditions WWI. hi ortier to Ol)tiAill an estinlat~c of the relative contributions of t,lte pigtti~~ttt, epithelium and the choroitl, respectively, to the experimental rcsult,s oltt,airtel i in Tal& 1 and IV, these layers were crudely W]JiI'.at~Cti according to tltc ]troce(iurrs out,lintxcl it1 the Methods. Table V shows t,lte results oltt,ainetl. In the lJullfrogs. the activit)irs of tlte Na 1-B’ and H(Y); ATPasrs iarc’ very similar in t’lte t\vo fritct,icJtts att(1 of tltch S;IIII(:
ntagnitutie as those in the pigment epitheliun~cltorc~id preparation. Thtl XJg” dTPase is also similar to that of the initial preparation but appears to Itre’lonrirtatt~ slightly in the choroid fraction. In the ra]Jbit. where separation of t htt fractions appeared to be more contplete, there is a clear tiiscriit~ittation Idxef~1i t,hc r.tqt~c:ti\-c enzyme activit’ies in the two fractions. The Na l-k’+ ATPase is higher itt t,lte q~it~ltc~lial cells than in the whole tissue homogenates and appears to be al)scttt Prottt tltr: choroicl fraction. C!onversely. activities of the Mg’ and HClO; ATPases itt t,he epitheli;tI ct~lls are lower than those of the whole 1totttclgSrnate and are only IO- W”,, of t,ltc It~v(~li itt the choroid. 4. Discussion The intent of this study was to tietermine whether the ion tratts])ort systcsttls postulated to account for active ion fluxes itteasureci across the short-circuited pigment epitheliun-choroid preparations are supported by ion-dependent ATPases (Lasansky and DeFisch. 1966; Miller and Steinberg, 1977a). Movement of sodium and potassium against electrochemical gradients is known to be driven by means of
ATPASES
IN
PIGMENT
EPITHELIVM
AXD
C’HOROID
4.53
energy derived from ATP via the ouabain-sensitive Na+-Kf ATPase (Skou, 1965). Whether bicarbonate and chloride are transported by means of similar, specific iondepen(lent ATPases is a matter of some dispute (van Amelsvoort. de Pont, Stols and Bontinp, 1977), but the HCO;-dependent enzyme is known to occur in several tissues in which active HCO; fluxes have been est,ahlished (Kasbekar and Durbin. 1965; Kinne-Saffran and Kinne. 1974; Riley. 19i7). In bhe combined pigment epitheliunl-choroid preparations from a.11species studied, significant Nat-K’ and HCO; ATPase activities are observtd. These activities might lje affected by the presence of either red blood cells or rod outer segments, the t’H.0 major cellular contaminants noted by Berman: Schwell and Feeney (1974) in their prepamtions of pigment epithelial cells brushed from bovine eyes. However, reel 1~100~~cells, while clearly present in our frog and rabbit preparations, are shown to have insignificant Mg 2+, Nat-K-1 and HCO; -ATPase activities in our tissue homogenates. Furthermore, rod outer segment,s appeared to be virtually absent in our hist,ological sections and in our suspensions of pigment el)itlleliun1-choroid material (suggest’ing possible differences in the ease with whic:h the pigment epithelium is srpamtetl from rod outer segments in different species). In any case. rocl outer sagmerits prol)ably contain no Na’-K-ATPase activit,y according to Zinmiernian. I)ac~nlt~ti and Bonting (1976) and P,erman. Azinrova a8nttGruba,kin (1977) who assayed t,his t*l~zyme activity in isolated photoreceptor cc11 fractions. Sitlc:fa the limiting cell niemln2ne for ion transport in t,lie pigment epitheliunchoroicl preparation resides in the continuous ta\,er of epithalial cells it has l,cten assunlctl that the active transport activities also rtlsi(le in this layer. Perhaps thr strongest evidence for this assumption comes fror11 t’hc, intratcellr~lar recortlings from pigm(lnt clpithelial cells (Miller and Steinberg. l%‘Th), from the observation that oUal,ili?l iq effective onlv at, the apical surface of these cells (Miller and Steinhcrp. 19iia). and frown the eke&s of iodate on the pigment epibhrliunl and transport of amincb ;tci(ls into and out, of t,he eye (Noel], 1963: Hecltly, (‘hakrapani a,ntl Litn. 1’37~). 1Yl’e11;1 vc attempted to determine whet’her the ion-tlelJetltlrt]t, .-1TPasc~ activities ;\r(’ Iocalizt~cl predo~~tinantly within bhis cell twycl. The isolatcatl pigment cl~ith~~liim~ fracticln \vhich we obtain prior to homogenization contained mostly intact cells itntt some t’r~ct pigment granules. but few other tissue fra,gnlents. The choroitl fraaticnl. 11~co~lt~rast. appeared to bc a very mixetl populat’ion of remaining pigment cpitheliat ctalls (c~y,ociallv hullfrogs), ret1 blood cells. blood vessels aiitt support~ing st,romitt illatrix. It1 t,htb ri~l)t)it.the enzyme activities of the t,wo fraction-: itre clearly clifferetrt. N:L K * ATPast: Ibeing restricted to the epithelial cells while \I$- and H(Y); ATl’asc~ :ire l)re(lonliuiint (the latter almost’ totally) in the choroid fract,ion. In the frog. t.hc rj)ithelial cells contain significant activities of bot8h the Na K- a,ntl HCO- ATPases. I)ut w lrethcr thrb choroitl is? likth t,he rabbit. tlevoid of the N;h -Ii- chnzynli is (lifEcult to detchrurine because of the greater contamination (If the choroicl with epithetiat cclts in thc~ frog preparations. Despite t.his dificnlt,y. it CatI 1beconcluded that the epithelial cells. Iloth of t’he bullfrog ant1 the rabbit. cont,ain an active Na --K- -stitnulatecl ATPastA. Moreover, in the bullfrog, this cell layer also contains a significant H(Y); Stillllllil.ttYl ATI’ase activity. but in the ral)l~it the activitv in thr fq~ithetii~t fracticm is to\\. lllilkillg its origin uncertain. Ilt~a~ving aside the question of the precise location of these enzyme activities. \vhioh is I)ryc~ntt the scope of the present paper, it is of interest to conlpare the specific a&vit’,v of t’he Na+-Ki- ATPasr in the isolated pigment epithelium fraction from the l,ullfrc,~ with the net flux of sottiunt across this cell layer reported in the recent work
These calcnlations of Na -K + ATPaw activit,! arc al~l~lical~le onI!+ under calcirttnfree conditiorls. since it was shown that t,he presence of 2 ttiY1 calciutti cot7rpletel~ inhibited this enzynle in hotnogenates Of pignlent, eltitheli77t77. This cellular larw is t#herefore sirnilwr to nlan,v other tissue.:: ( hJIlthg. tiitnon at7tl Hawkins, 1961). it&ln(ling the retina (Winkler and Riley. 1977). where calci77ttt iOilS were previously showt to it7hilA ?;a .--Ii ATPase. The inhiltitory effect of calciuttt on this ctnzyttw it7 t,lre pigntent epitheliunt would appwr to Ite of partic77far int(erest since the intracellular concent~ration uf CakillIll iI1 t’his Cell is retNJI%ed to Ibe in die Illd~iI1l~JlW railge (Hess. 1975). This high concent,ration woul(l clcarl!, result in ~ulwtantial itthi hitiott of the Na-Bi ATPase. and wonld seen1 to preclude any futlctiottwl wlationship Iwtween Nit j-B+ ATPase and sodiutt7 t~ransport. It, Itlust,. thcrefow. he assuructl that virt,ttallv all of this calciutn is bo7tnd \&hit7 the Itignlent grattrtlr;. 117lWV~~ilitl~l~~for inhit~itory ittt,eracCion with this enzwne. A siniilar cluantitative argunient co17ltl he niatle relating the actjivity of the H( ‘O,stitnulatetl ATPase and the active tra.tqort of hica~rbonate postulatetl in the lt7tllfroy t(J acconnt for the discrepancy between Ilet ion fluxes of sodiutii atitt chloride atttl the short circuit current. However. t,he H(‘O~ -sbimulated ATPase is not cntirelv ion specific, nor is it clear whether the enzvnte is sit77nted on die ptasnla ttleinbratics of t,he cells in which it is founcl. Such a location is a prereq77isite for transcellular transport of any ion Ity t#he enzytne. Until this point is resolved ant1 direct, tneas77wtt7entSs of bicarbonate flax are ohtainctl the tlettlonstration of a q77antitative eq77ivalence between enzymatic activity and transport is not as compelling as for t,he socliuttt iwt . Indeed, in the rabbit. there is no significant hicarlmnate-stin77datetl ATPase activity in the pigment epit~heliuni. In summary. t,he enzyme a&dies of the pigment epithelium appear to be capable of tnediating the active transport of sodium ions and possible of bicarbonat8c ions. To date, there is no infornlation WI the q77est’ion of ltossil& tnechanisnw for the active transport of chloride and ot,her ions such as calciutn which also participate in the overall ion fluxes across the pigment, el~it~heliun~~choroid.
This work was supported in part by (+rattts El’ 00541 and EY 01219 front the National Eye Institute, I-nited States Public Health Service. The authors thank 3Iiss Penny Agnew, an undergraduate student at Oakland University, for histological preparations. The a77thors also thank Dr Charles Lindernann and Mr Ian Fentie of the Departrnent of Biological Sciences for photographing our preparations it7 their Reichert Zetopan microscope. The secretarial assistance of MS Dawn Bocbel is very much appreciated. REFElXENCES Berman, A. L.. Azimova, A. M. and Gribakin. F. G. (1977). Localizat’ion of Ss+-K+-4TPase and Ca”+-activated Mg”+-dependent ATPase in ret’inal rods. T’is. Res. 17, 527-36. Berman, E. R. (1971). Acid hydrolases of the retinal pigment epithelium. Inuesf. Ophthalmol. 10, 64X.
ATT’ASES
IS
PIGMERT
EPITHELIUM
AND
CHOROID
Berman. E. R., S~hwell, H. and Feeney, L. (1974). The retinal pigment epithclium. composition and structure. 1?lzxsf. O~hthnlmol. 13, 675-87. Bonting, S. L., Simon. K. A. and Hawkins, N. JI. (1961). Studies on sodiu~n-potassirllu adenosiw triphosphatase. I. Quantitative distribution in several tissues of the Biochem. Biophys. 95, 416-S. Fiske, (‘. H. and Subb arow. 1--. (19%). The calorimetric d~~termiIlatior1 of phosphorus. C’hrmn.. 66,
&it5 Chemiwl activated cat. dwic. 6. 11601.
375-400.
(:locklin. V. C. and Potts. A. 31. (196.5). The metabolism of retinal pigment cell cpitheliiini. 11. Respiratiou and glycolysis. r’~wt. Ophthnlnrol. 4, 2%~34. Hess, f-l. H. (1975). The high calcium content of retinal pigment epit,ht hum. Eq. Eye Hex. 21, 471 9. Kasbekar. 1). K. and Durbin. R’. P. (1965). .\n adenosine triphosphataso from frog gastric mi~cosa. Biorhinr. Biophys. Acfn 105, 47”%u. Kinno-Suffran. E. and Kinne. R. (1974). Presence of bi~art)orlatr-stilllulatrd ;\TPase in the brush bordtr nlicrovillus membranes of the proximal tubriles. /‘,or. Sot. Esll. Biol. (S. F.) 146, $51 -3. Lasansk~-. A. and DeFisch. F. \V. (1966). Potential, current and ionir Huses across the isolntetl retinal pigment epithelium and choroid. d. C:P/Z. PA,+sio/. 49, 919-2-t. Jlillcr. S. S. and Steinberg, R. H. (1976). Transport, of tnurine. r,-methionine and :;-O-methyl-l, ylu~~osc across frog retinal pigment epithelium. 6xp. 6!/r fies. 23, 177789. ?tIiller. S. S. and St)einberg, R. H. (1977a). Active transport of ions across frog retinal pigultwt cpithelitim. 1i.r~. Eye Res. 25, Zl.538. Jlillcr. S. S. and Steinberg, R. H. (1977b). P assire ionic propertic~s of frog pigment epitholiulil. .J. .Il~,tr. Hiol. 36, 337-72. Xocll. \\‘. 1~. (1952). Bzide sensitive potential ditfrrencw across the eyv bulb. ilw!. .I. Physiol. 170, “17-38. Soell. \V. Ii. (19611). Cellular physiology of the retina. J. Opt. Sot. A/T/er. 53, 36-M. Socll. \\‘. K., Crapper, D. R. and Paganelli, C. V. (1965). Transretinal currents and ion tlusw. In ?‘rtrn.scelluZnr Xenrbrrtne Pofrnfinls md lowic Flwws (Eds Snell. F. and Pu’oell, \V. I~.). Nc\\ York: C:ordon and Breach. Redtly. V. S., Cliakrapani, 13. and Lini, C. P. (1977). Rlooll-vitreous barrier to amino acids. 6x/l. Eye RPS. 25, 543-54. Riley. 31. V. (1977). Anion-sensitirc ATPase in rabbit cornea1 endothelium and its relation to cwneal hydration. Ex~I. Eye Rea. 25, 483-95. Hkou, .I. (1. (1965). Enzymatjic basis for active transport of Sa+ and 1~ f across ~11 membrane. l’h,ysiol. Rer. 45, 5966617. Steinbwg, R. H. and Miller, S. S. (1973). Aspects of elrc,trolyte transport in frog pigment epitlu!lium. Eq. Eye Res. 16, 365 -72. ran Amelsvoort, J. $1. >I., de Pont. J. J. H. H. AI., Stols, A. L. H. and Bonting, S. L. (1977). l-: there a plasma-membrane-located anion-sensitive ,1TPasc? IT. Further studies on rabhit kidney. Biochew. Biophys. ilctu. 471, 78-91. \Vinklw. 1% S. alid Riley, $1. V. (1977). Sal-K+ and HCO; ,\TPase in retina: dependence on cal~iutn and sodium. Iwest. Ophthnlw~ol. 16, 1151-4. Youtig. I’,. \V. and Rok. D. (1969). Participation of the retinal pigment epithelium in the rod out,er svgnient renewal process. .I. Cell Biol. 42, 392403. Zimnwman. I\‘. F.. Daemen, F. J. RI. and Bonting, S. I,. (1976). Distribut,ion of enzyme activities itr subcellular fractions of bovine retina. J. Biol. Churn. 251. 4700-06.
ATPase Activities in Retinal Pigment Epithelium and Choroid
The Ilg”--. the Sal--K&and the HCO;-dependent i\TPase activities of retinal pigment epithelinln~choroid tissue from grassfrog, bullfrog and rabbit, have been measured. In the frogs, X’Ig”?~ ATPase activity was het,neen 1 and 2 ~~mols Pi liberated hr-l rng--l protein, while Sn-l--K+ ATPase and HCO, ATPase were about PO and %“,, of this activit,p. respectively. Corresponding values for the mbbit were X.5 pm01 Pi hr--’ mgpl prot’ein. HO,, and WC,. Sal ~.K+ ATPast, activity in each species \vas completely inhibited b>l 2 mll-calcium. ~vhilr H(K1, ATPase was llnaffected by calcium or hy ouahain. The pigment epithelium-choroid preparations of bollfrog and rabbit were scraped to yield suspensions of isolated epithelial cells. relatively free of other t,isstle component,s: and and red blood a residual fraction which contain4 some’ rpit helial cells. c~horoidal elements ~4ls. >Ya- -Ii+ ATPasa activity was present in t,he epithelial culls of both species and absent from the choroid of the rabbit. H(‘OJ XTPnse and IrIg’ 1 ;\TE’nse activities were ronghlg bl(t in thr rahhit xverr concentrated in the equally dist,rihllted in the hnllfro, u fractions. choroid. The result,s are discussed in relation to the postulated active ion transport pr~xcsse~ of the pigment epithelium. h-p!/ forces: pigment epithelium ; choroid; ac+ivc trallxport : >I$‘~ .ZTPase; SR’ -I< .\Tl’asc; HCO, AITPasr; orlabain : cal(Gllm.
1. Introduction The pignlent, cpitheliuni performs a nninher of functions which xe essential for the survivd of the photoreceptor cell and for the rneint,~?na.nce of retinal responsivrwess to light.. TINS. the pigment epit,heliultl hell’s to control t,he lengtll and shap of the outer segment of’ the photoreceptor cell 1)~ R conq~les process of phagocytosis of the tliStil.1 til)s of thcl outer segment discs (Young itilt JZol<, 1969) and it regubtes the flow of inolecnles and ions hetwwn t,lie choroidal hlooti ant1 t’he neural retim (Socll. 1952. 1963; Lassnsky and DcPisoh. 1966; Steinlwrg ant1 Miller. 1973: Niller and Steinlwrg. 19i6. 19’iia.l)). IOIl fnscs across isolated pjgnmit cpit’heliunl -choroit’l ~)rqssmtiutis have heen nieasu r-et1 in the itlweuce of an elect~rrxhenlic;%l ]wtcIltial t~ifftwnce in order ix delmxiine which ions are actively ~r~tllSJ)~Jrtcd by the pignicnt~ tyit~tielilln~. A net transfer of chloricle froni t’he retina to t’lie choroitl ztnd a net t.miisfer of sodiuu1 from the choroid to the retina \i-ere found in the tmd (B’clfo ~r~~~irrus) :mtl ltullfroy (RUM c:trtesbeinm) prqmmtions (l~;tsansl<>~ ant1 I>eFisch. 1966; Miller and Stein1~er.g. 197’i;i.). In hot11 thrw slwcies the twm short circuit, current could not Iw ent,ircI!s
?Lccouutecl
for
fiv
the
nrht. fluxes
of srrdiurll
~(1
chlorj(le
a.ntl
it. WAS suggest,4
that, at1 active H(‘O;/H-+ transport s,vstenl Inay contAMe bo this current. In contrast, to tlicsih studies 011 colrl~t&~otletl verte\)mtes. Noel1 (1963) xntl Nnell. C’rqq)er ant1 Paparlc~lli (1965) found evidence of a,n active transport of potassium from bhc choroitl t,c)\varti thc& vitreous huxnor which a,ccowt,etl for a major fraction of the short circnitv curvent across the Ijignlent epitheliunl in the ral)l)it. That ount)ain, when added to t.he apical (rebinal) side of the isolated pigment, epit’heliulu-choroicl preparation of t,he frog. a,lters the net flux of sodiunl across the pigment, epit’helium (Miller and Steinberg.
2. Materials
and Methods
C+rassfrogs (Rtow f)iljie~ln) a1tc1 bullfrogs (I/(ol(( wfwhf~i~~)~~6) were 11;t1,1; ;t(laptecl for sc\-em1 hours and decapitated. The eyes were removetl, bisected equatoriall~mid the> WJII hiued retina-choroitl n-as e;~rcfull~- rlissect,etJ away frort~ the scleru. The IEUIX~ retimr GIS t,hett gently clet.;whetl from t,he remaining JGgment epitheliulll choroicl tissue. This procetlure is similar to that nsecl bv T,aruttskv nntl DeFisch (1966) ant1 Miller ;r~ul Steittbrr,g (19771)) for obtaitiitlg the pigmertt epitheliu;Il choroitl free from other ocular tissues. I’ignletttetl rabbits were killecl by ;tir etnbolistll i~ttd the eyes \\‘ere ettwleatetl. Tlrra posterior part of the eye was freed c~f vitreous humor x~ittcl cut iJJt,o li;llves through the optic tterre. The neural retitiit tloate(l front t,lie retrraitiitlg tissue after :L short time it1 1tor11ts1 buffered saline. The pigtncrlt epitl~elint~l~c~l~or(~i~l wits then separ;itetl t’ro~r~ t,hr ac,Jtw by severiug the blood veswJs \vitlt FiskeforceJ)s ~ritd liftiiig ofi the isolated J”.V~~ilJ’~l~iOll. Tlte iigment epitheliunt -choroid frottt cac11 of the species was hotnogertizetl it1 water, usitig it, glaw pestle atid t,ul,c. W;r.trr \ws choseit its the Iiunrc~gei~iziiip medium tlcspite t,he ,liSilllV;lllt;t~e of aggreg;itioJt of cell c~~~rirpot~eiit:Y sometitiiw observed with this ~~rowclure (\\ Iiic,lJ triight, il~COUllt for the r;iriiition iii &T-it! htrl ottp pJ~epitr~tti011 to ;ttlc>tlleJ~). 1‘llt~ a~clv;iJititge Of ~~~ilte~ \IibS tllilt the OsttiOt~i( shock lvsetl cells ;ttirl clisperswi JGgnwtlt gr;liiules. givinfi it higher specific, activity th;ttl Jlotlto,~ett;tt,es in \~ufkr. without twcwt 10 .~h~~ltll)ilia;ttio~~” wit,11 detergents. Tissue front tn.0 ,grwsfrog or bullfrog evt~s \V;LS uwl for 2 1111 of hotnogetiate yielding itui it\-rra,ge protein couc~ettt,r;ttic~r~ of 0.4.; ttig: ‘Itrtl for* gtxssfro,g ;r11t1l.tt ntp/~ul for billfrog. For t,lre r;rhhit, tissue frollt otle half of:tlJ eve \V;LS hOlll~J~etLiZ~~~~ jli .I ml of wter , giving itn :L\‘erxge protein cottc~rtttr;itiotl of 0.41 tltgitttl. .\ crtttlc sep;rr;~tiott of the IJigt1Jeitt rpitdteliuttk -cltoroi~l ittto its c~c)llJlmtleltt ]kart.q \vils ;~lso ;~Ctemptetl. After t,lic wtltbinetl prcJxtr:itioti w i 1+ isol;rtrtl frotll the s(‘Ier:L at)11 treUJ’;l~l rctili;t it \YitS phcetl witli ;I, fe\v (lrops of J~hvsiol~~~ic;~J wlinc 011 i, fht surface untlct~ :I dissec,t,itig tnicrosaope. L\ sp;l,ttil;t \viiq use11 to push c,luttrJw of pigtnctttj epit~lielial (*ells frotii t,hca ttt~clerlyiug choroid. C’ells cattle free itt sheets. stltitll grouJx6 ittt(1 seJxtx;ltely, but f’e\\free l)iptetlt grandes wew ohrn~etl. ittclic,;ttittg th;tt few cells \vcre brl~keii /)I’ this J)t’Ouetliiw. :In alternativr tnethotl. hlretlt,I\r sh;tkill,c tlte tissue itt it st11;11l t,ot.tlr (.otttainitt~ s;ilitict itiicl several KlitSS I,e;r,ds, resultcat! iii fair rrlllov~ll of cells front t)llr choroid;tl tissue but excessive dispersion of pigitietit gr;itmles, supgestitrg that contents I,oth of epithelinl c~,lls :1,t1(1c.ells from the choroitl were included iti the suspension. After scrapittg, t IlC isol;ttwL c.ells were removetl with t)lie c)verl,viilg flui, ;~lttl hnniogettizetl :11ttJtlilutc>cJ :tppronxs also hotrtogenize~l itt s;rlitlcl. pri;ttely. The retnailtillg t,issuc, clesigtlatetl “choroi~l”, III tlie caqe of the bullfrog, this tissue wit,s still hearily piglrtetltetl, p:irtlv l)>- rcittaittittg epiBJielia1 cells (estiiiiat,ed 3s 20’: I1 of total) iLll(l bv t,he pigtuellt stirroutitlitig tile bl0d ve,qsels (see Fig. 1). The rabbit rhuroid. having 110 pigmetlt a11(1 lvitq Hitt on tltc’ scJrr;t. ~~nltl 11e scrap&l virtually free of epitlielial cells.
.ITPxse activity was measured by- determiuing the phosphate according to the method of Fiske and Subbarow (1925), as modified
liberated from ATP bv Winkler and Riley
ATI’ASES
IS
PIGRIEKT
EPITHELIl’~l
hS1)
(‘HOROID
117
(1977). The incubation medium contained Tris-HCl, 50 111~; NaC!l, 120 mx; KC& 5 111~; ATP (Sigma Chemical Co., St. Louis, MO.) 2 nm: MgSO,, 2 rnx; awl EDTA, 0.3 HIM. OuabitiIr. when present, was 0.1 nm. Sodium bicarbonate (‘25 nm) was substituted for all eyuivnlent amount of NaCl when HCO; ATPase was determined. The term HC’O; hTP;w is used itlstead of aniorl-stusitive ATPnse bemuse bicmbona.te is the only IN:~~IU ;wion ~lther thm chloride fouud in intro- amI extrircellular fluids. However, t,he enzyrlw activity of the pigment epitheliun-choroid preparation was also stimulated by wltit~~ ;t~r(l zl11~et1 minor variations when other anions or sucrose were substituted for chloritk. pH \v:I.~ brought to 7.7 for frog esperiulents and 7.4 for rabbit experimeutn. Althcqgll 2.5 n111 NaHCO, is not, compatible with these pH values without bubbling with an appropr.iate tmrtial pwssure of CO?, it was observed that such bubbling did not change thl* a(,ti\-it\ of the preparations. It was concluded that significant loss of C’O, from the nlelliuul tlill Ilclt Iwwr during the period of illcubation. .\lic~not.x of homogenate of 0.1 1111were used in final irssny volumes of 0.5 ml. triplic-xtt. srta ol’t ulws being used for ewh set of conditions for a given homogenate. Frog prepar;Lticlw ~ve~‘t~ illcxhhetl at 22°C for 40 min. rxhbit preparatiolls at 37°C for 30 min. The extent ot ATI’ It!xlrolyis was less than iLO’),, and was linear with time below this value. It \Y;I~ foulIll ttmt the specific activity of a given preparatiou declined by ~8I’:(, over a fourfc&l r:r~il~:l~ 01’ proteiri c~oiicentratiou in the assay medium, but’ wit’hiri ;I single species the v;lri:lticlti of protein in honiopenates wiis ilever greater th;lii t\rcbfr,ld.
3. Results
VII* ;:lqmtra~~~ of the pigment el’itheliunl-chor(li(l lml~aration cbl)titiuetl f’rollr the I)ulli’ro~ is show) in Fig. 1. The epithelial cells. most hearilp pigmented towmls t,llca apica, Glcl. lie in a single uniform layer across t’he yltrfacc of the tissue. support,wl II\T
‘l’tl(l isolated epithelial cells of the hullfrog are shown in Fig. 2. It, alq)ears t,llil,t ;I. sing11~ cell Iaver is present, the cellsprobahl\being held together 1)~ Bruch‘s mc~nl)r;~m~. Ilnt it. is tlclt certain whether parts of tie choriocapillaris arc ‘Ml attachctl ~IXIIII Fig. 1 this wonld appear to be the most likely site of cleavage. The free cells are clra,~l! clev~ li(l of ot#her tissue components. Few red I)lootl wlls antI few free pignwntj gr;~lnll<~~ ww
a ~qmreiit~
in these
suspensions.
nit> specific mtivities. based ou protein collceiltri-ltion. of the &I$ -- ant1 iTi1 1-K st,illllll;ltetl ATl’ases of the pigment epithrrli7llll~c.floroi~l frank t,he three espcri~lwntai mill& we sh~~wn in Table I. The colcl-~~lwcled auinmls showtl low R4g’j ATPaw activitiw and when ouahain was omitted from the awav medium there was a sltlall iin(I +nificant increase in the rate of ATP h+)l+s ‘indicat’ing the presence of a the activit’y of t)his enz,vme was about lV)b of t’ht, Ka I< ATPase. In the bullfrog. Mg’: .ZTPase xctivitv and in the grassfrog about 200,,. The specific activities of t,lw &tg”! ;7nrl h’a-’ -K+ ATPases in the pigmmtetl ralbbit w,re alqmsinmtel~~ six- amI
variability from experiment to exl~eriment in the Mg 2+ ATPase activity, the ortGsion of ouahain always led to an increase in t,he rate of ATP hydrolysis. This increase in ATPase activity (Na+-K b ATPase in Table II) was shown to he significant using the method of t#esting the significance of the mean of differences betwcen the Mg2+ and iMgz+ +Na+-Kf ATPase values for each experiment; this procedure was justified
ATPASES
Frc after cells.
IN
PIC:.\IENT
EPITHELIIJM
AND
CHOKOID
4.ilI
11. \‘.
1:ILk;s
lq:‘I‘ .\I.
since each pair of values was obtained front. a single hon~ogenattt. This tnethotl is particularly useful for denlonstrat,ing the significance of small tliffcrencw whrtt variability between individual preparations is of a magnitude cotnparal~lo to t,lte crl)servcd changes in AT&se activky when mat)ain is omitted. This is I twst. iLl)l)a~rt~I~t in the case of the Imllfrog when a sinlple conq)arison of the differelm of the nLeau5 leads to a P value hy Stujent’s t-test’ of appxiinately 0.1. T1irrrfi.m. we have: t~sctl t,lic mean of t)lie tlifferenccs in t,he statistical atmlyses throughout. Table IIT. giving results of c~xprinrents in the presence of 2 tt~hi calcirm. Aows that8 this ion c~Jlll#!te~y inhihitr; the Na~,--K ATPase activity of’ the Ijnllfrog l)rt’l)arat,iun. Again. i\Ig”-~ ATIke a,ct,ivity varietl twtwen homogcnatc-. Imt in no caw
“43lI 1.61 14s I.05 14;i
II.91
(I..34 Il.76 (I.,? I I I.!%
-‘.S4 I ,iT 2.1-i 1.21’ I.filI 1.tl.i I kill II.!)-1 Il.;? I.12
452
)I.
\‘.
RILEY
K’J‘ AI.
Homogenates of tSltr cottthincci pigttteub c]tit,he]iutn--cltoroill anti of t tttt sca]“tt’it tcstl fractions (SW below) all cotttainetl 4pnificattt atttount,s of l11011tl.To etitttittatv t 111, ])ossibilitv that this co~q~onerit of t,llc hOttiOgwat~cs cOith’i~lllt~?~~ sltil~tiLtitid1\’ 10 ittl\ of t)he nteasured ATPase activities. qu\])(Gotts of’ frog an(i ritl~l)it I~loc~tl.(‘ont:linitl< rttuclt higher cottccnt~rations of reel I)lootl cells t’hatt in the t,issut, ]~~t~l~;t.t~atic~t~~. \\(‘I’(’ also assayed. The l\;lgz* ATPast! act#ivity of frog t)too(i \\-its 0.15 ptt101 I’i’ltr/tlt,ti probein (average of five experiments). anti of rabbit8 l~looci was 04 ~ntol/ttr;‘nrg protein (average of four experiments). an(l the Xa- K - and H(Y); .\‘I’Pnsr a(+tivitif,q were negligible under the conditions WWI. hi ortier to Ol)tiAill an estinlat~c of the relative contributions of t,lte pigtti~~ttt, epithelium and the choroitl, respectively, to the experimental rcsult,s oltt,airtel i in Tal& 1 and IV, these layers were crudely W]JiI'.at~Cti according to tltc ]troce(iurrs out,lintxcl it1 the Methods. Table V shows t,lte results oltt,ainetl. In the lJullfrogs. the activit)irs of tlte Na 1-B’ and H(Y); ATPasrs iarc’ very similar in t’lte t\vo fritct,icJtts att(1 of tltch S;IIII(:
ntagnitutie as those in the pigment epitheliun~cltorc~id preparation. Thtl XJg” dTPase is also similar to that of the initial preparation but appears to Itre’lonrirtatt~ slightly in the choroid fraction. In the ra]Jbit. where separation of t htt fractions appeared to be more contplete, there is a clear tiiscriit~ittation Idxef~1i t,hc r.tqt~c:ti\-c enzyme activit’ies in the two fractions. The Na l-k’+ ATPase is higher itt t,lte q~it~ltc~lial cells than in the whole tissue homogenates and appears to be al)scttt Prottt tltr: choroicl fraction. C!onversely. activities of the Mg’ and HClO; ATPases itt t,he epitheli;tI ct~lls are lower than those of the whole 1totttclgSrnate and are only IO- W”,, of t,ltc It~v(~li itt the choroid. 4. Discussion The intent of this study was to tietermine whether the ion tratts])ort systcsttls postulated to account for active ion fluxes itteasureci across the short-circuited pigment epitheliun-choroid preparations are supported by ion-dependent ATPases (Lasansky and DeFisch. 1966; Miller and Steinberg, 1977a). Movement of sodium and potassium against electrochemical gradients is known to be driven by means of
ATPASES
IN
PIGMENT
EPITHELIVM
AXD
C’HOROID
4.53
energy derived from ATP via the ouabain-sensitive Na+-Kf ATPase (Skou, 1965). Whether bicarbonate and chloride are transported by means of similar, specific iondepen(lent ATPases is a matter of some dispute (van Amelsvoort. de Pont, Stols and Bontinp, 1977), but the HCO;-dependent enzyme is known to occur in several tissues in which active HCO; fluxes have been est,ahlished (Kasbekar and Durbin. 1965; Kinne-Saffran and Kinne. 1974; Riley. 19i7). In bhe combined pigment epitheliunl-choroid preparations from a.11species studied, significant Nat-K’ and HCO; ATPase activities are observtd. These activities might lje affected by the presence of either red blood cells or rod outer segments, the t’H.0 major cellular contaminants noted by Berman: Schwell and Feeney (1974) in their prepamtions of pigment epithelial cells brushed from bovine eyes. However, reel 1~100~~cells, while clearly present in our frog and rabbit preparations, are shown to have insignificant Mg 2+, Nat-K-1 and HCO; -ATPase activities in our tissue homogenates. Furthermore, rod outer segment,s appeared to be virtually absent in our hist,ological sections and in our suspensions of pigment el)itlleliun1-choroid material (suggest’ing possible differences in the ease with whic:h the pigment epithelium is srpamtetl from rod outer segments in different species). In any case. rocl outer sagmerits prol)ably contain no Na’-K-ATPase activit,y according to Zinmiernian. I)ac~nlt~ti and Bonting (1976) and P,erman. Azinrova a8nttGruba,kin (1977) who assayed t,his t*l~zyme activity in isolated photoreceptor cc11 fractions. Sitlc:fa the limiting cell niemln2ne for ion transport in t,lie pigment epitheliunchoroicl preparation resides in the continuous ta\,er of epithalial cells it has l,cten assunlctl that the active transport activities also rtlsi(le in this layer. Perhaps thr strongest evidence for this assumption comes fror11 t’hc, intratcellr~lar recortlings from pigm(lnt clpithelial cells (Miller and Steinberg. l%‘Th), from the observation that oUal,ili?l iq effective onlv at, the apical surface of these cells (Miller and Steinhcrp. 19iia). and frown the eke&s of iodate on the pigment epibhrliunl and transport of amincb ;tci(ls into and out, of t,he eye (Noel], 1963: Hecltly, (‘hakrapani a,ntl Litn. 1’37~). 1Yl’e11;1 vc attempted to determine whet’her the ion-tlelJetltlrt]t, .-1TPasc~ activities ;\r(’ Iocalizt~cl predo~~tinantly within bhis cell twycl. The isolatcatl pigment cl~ith~~liim~ fracticln \vhich we obtain prior to homogenization contained mostly intact cells itntt some t’r~ct pigment granules. but few other tissue fra,gnlents. The choroitl fraaticnl. 11~co~lt~rast. appeared to bc a very mixetl populat’ion of remaining pigment cpitheliat ctalls (c~y,ociallv hullfrogs), ret1 blood cells. blood vessels aiitt support~ing st,romitt illatrix. It1 t,htb ri~l)t)it.the enzyme activities of the t,wo fraction-: itre clearly clifferetrt. N:L K * ATPast: Ibeing restricted to the epithelial cells while \I$- and H(Y); ATl’asc~ :ire l)re(lonliuiint (the latter almost’ totally) in the choroid fract,ion. In the frog. t.hc rj)ithelial cells contain significant activities of bot8h the Na K- a,ntl HCO- ATPases. I)ut w lrethcr thrb choroitl is? likth t,he rabbit. tlevoid of the N;h -Ii- chnzynli is (lifEcult to detchrurine because of the greater contamination (If the choroicl with epithetiat cclts in thc~ frog preparations. Despite t.his dificnlt,y. it CatI 1beconcluded that the epithelial cells. Iloth of t’he bullfrog ant1 the rabbit. cont,ain an active Na --K- -stitnulatecl ATPastA. Moreover, in the bullfrog, this cell layer also contains a significant H(Y); Stillllllil.ttYl ATI’ase activity. but in the ral)l~it the activitv in thr fq~ithetii~t fracticm is to\\. lllilkillg its origin uncertain. Ilt~a~ving aside the question of the precise location of these enzyme activities. \vhioh is I)ryc~ntt the scope of the present paper, it is of interest to conlpare the specific a&vit’,v of t’he Na+-Ki- ATPasr in the isolated pigment epithelium fraction from the l,ullfrc,~ with the net flux of sottiunt across this cell layer reported in the recent work
These calcnlations of Na -K + ATPaw activit,! arc al~l~lical~le onI!+ under calcirttnfree conditiorls. since it was shown that t,he presence of 2 ttiY1 calciutti cot7rpletel~ inhibited this enzynle in hotnogenates Of pignlent, eltitheli77t77. This cellular larw is t#herefore sirnilwr to nlan,v other tissue.:: ( hJIlthg. tiitnon at7tl Hawkins, 1961). it&ln(ling the retina (Winkler and Riley. 1977). where calci77ttt iOilS were previously showt to it7hilA ?;a .--Ii ATPase. The inhiltitory effect of calciuttt on this ctnzyttw it7 t,lre pigntent epitheliunt would appwr to Ite of partic77far int(erest since the intracellular concent~ration uf CakillIll iI1 t’his Cell is retNJI%ed to Ibe in die Illd~iI1l~JlW railge (Hess. 1975). This high concent,ration woul(l clcarl!, result in ~ulwtantial itthi hitiott of the Na-Bi ATPase. and wonld seen1 to preclude any futlctiottwl wlationship Iwtween Nit j-B+ ATPase and sodiutt7 t~ransport. It, Itlust,. thcrefow. he assuructl that virt,ttallv all of this calciutn is bo7tnd \&hit7 the Itignlent grattrtlr;. 117lWV~~ilitl~l~~for inhit~itory ittt,eracCion with this enzwne. A siniilar cluantitative argunient co17ltl he niatle relating the actjivity of the H( ‘O,stitnulatetl ATPase and the active tra.tqort of hica~rbonate postulatetl in the lt7tllfroy t(J acconnt for the discrepancy between Ilet ion fluxes of sodiutii atitt chloride atttl the short circuit current. However. t,he H(‘O~ -sbimulated ATPase is not cntirelv ion specific, nor is it clear whether the enzvnte is sit77nted on die ptasnla ttleinbratics of t,he cells in which it is founcl. Such a location is a prereq77isite for transcellular transport of any ion Ity t#he enzytne. Until this point is resolved ant1 direct, tneas77wtt7entSs of bicarbonate flax are ohtainctl the tlettlonstration of a q77antitative eq77ivalence between enzymatic activity and transport is not as compelling as for t,he socliuttt iwt . Indeed, in the rabbit. there is no significant hicarlmnate-stin77datetl ATPase activity in the pigment epit~heliuni. In summary. t,he enzyme a&dies of the pigment epithelium appear to be capable of tnediating the active transport of sodium ions and possible of bicarbonat8c ions. To date, there is no infornlation WI the q77est’ion of ltossil& tnechanisnw for the active transport of chloride and ot,her ions such as calciutn which also participate in the overall ion fluxes across the pigment, el~it~heliun~~choroid.
This work was supported in part by (+rattts El’ 00541 and EY 01219 front the National Eye Institute, I-nited States Public Health Service. The authors thank 3Iiss Penny Agnew, an undergraduate student at Oakland University, for histological preparations. The a77thors also thank Dr Charles Lindernann and Mr Ian Fentie of the Departrnent of Biological Sciences for photographing our preparations it7 their Reichert Zetopan microscope. The secretarial assistance of MS Dawn Bocbel is very much appreciated. REFElXENCES Berman, A. L.. Azimova, A. M. and Gribakin. F. G. (1977). Localizat’ion of Ss+-K+-4TPase and Ca”+-activated Mg”+-dependent ATPase in ret’inal rods. T’is. Res. 17, 527-36. Berman, E. R. (1971). Acid hydrolases of the retinal pigment epithelium. Inuesf. Ophthalmol. 10, 64X.
ATT’ASES
IS
PIGMERT
EPITHELIUM
AND
CHOROID
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(:locklin. V. C. and Potts. A. 31. (196.5). The metabolism of retinal pigment cell cpitheliiini. 11. Respiratiou and glycolysis. r’~wt. Ophthnlnrol. 4, 2%~34. Hess, f-l. H. (1975). The high calcium content of retinal pigment epit,ht hum. Eq. Eye Hex. 21, 471 9. Kasbekar. 1). K. and Durbin. R’. P. (1965). .\n adenosine triphosphataso from frog gastric mi~cosa. Biorhinr. Biophys. Acfn 105, 47”%u. Kinno-Suffran. E. and Kinne. R. (1974). Presence of bi~art)orlatr-stilllulatrd ;\TPase in the brush bordtr nlicrovillus membranes of the proximal tubriles. /‘,or. Sot. Esll. Biol. (S. F.) 146, $51 -3. Lasansk~-. A. and DeFisch. F. \V. (1966). Potential, current and ionir Huses across the isolntetl retinal pigment epithelium and choroid. d. C:P/Z. PA,+sio/. 49, 919-2-t. Jlillcr. S. S. and Steinberg, R. H. (1976). Transport, of tnurine. r,-methionine and :;-O-methyl-l, ylu~~osc across frog retinal pigment epithelium. 6xp. 6!/r fies. 23, 177789. ?tIiller. S. S. and St)einberg, R. H. (1977a). Active transport of ions across frog retinal pigultwt cpithelitim. 1i.r~. Eye Res. 25, Zl.538. Jlillcr. S. S. and Steinberg, R. H. (1977b). P assire ionic propertic~s of frog pigment epitholiulil. .J. .Il~,tr. Hiol. 36, 337-72. Xocll. \\‘. 1~. (1952). Bzide sensitive potential ditfrrencw across the eyv bulb. ilw!. .I. Physiol. 170, “17-38. Soell. \V. Ii. (19611). Cellular physiology of the retina. J. Opt. Sot. A/T/er. 53, 36-M. Socll. \\‘. K., Crapper, D. R. and Paganelli, C. V. (1965). Transretinal currents and ion tlusw. In ?‘rtrn.scelluZnr Xenrbrrtne Pofrnfinls md lowic Flwws (Eds Snell. F. and Pu’oell, \V. I~.). Nc\\ York: C:ordon and Breach. Redtly. V. S., Cliakrapani, 13. and Lini, C. P. (1977). Rlooll-vitreous barrier to amino acids. 6x/l. Eye RPS. 25, 543-54. Riley. 31. V. (1977). Anion-sensitirc ATPase in rabbit cornea1 endothelium and its relation to cwneal hydration. Ex~I. Eye Rea. 25, 483-95. Hkou, .I. (1. (1965). Enzymatjic basis for active transport of Sa+ and 1~ f across ~11 membrane. l’h,ysiol. Rer. 45, 5966617. Steinbwg, R. H. and Miller, S. S. (1973). Aspects of elrc,trolyte transport in frog pigment epitlu!lium. Eq. Eye Res. 16, 365 -72. ran Amelsvoort, J. $1. >I., de Pont. J. J. H. H. AI., Stols, A. L. H. and Bonting, S. L. (1977). l-: there a plasma-membrane-located anion-sensitive ,1TPasc? IT. Further studies on rabhit kidney. Biochew. Biophys. ilctu. 471, 78-91. \Vinklw. 1% S. alid Riley, $1. V. (1977). Sal-K+ and HCO; ,\TPase in retina: dependence on cal~iutn and sodium. Iwest. Ophthnlw~ol. 16, 1151-4. Youtig. I’,. \V. and Rok. D. (1969). Participation of the retinal pigment epithelium in the rod out,er svgnient renewal process. .I. Cell Biol. 42, 392403. Zimnwman. I\‘. F.. Daemen, F. J. RI. and Bonting, S. I,. (1976). Distribut,ion of enzyme activities itr subcellular fractions of bovine retina. J. Biol. Churn. 251. 4700-06.