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Active transport of ascorbic acid into lens epithelium of the rat
Exp. Eye Res. (1989) 49, 875885
Active Transport of Ascorbic Acid into Lens E p i t h e l i u m of the Rat* J O S E P t t DIMATTIO
Department of Physiology and Biophysics, New York University School of Medicine, 550 First Avenue, New York, NY 10016, U.S.A. and New York Veterans Administration Hospitalt, 408 First Avenue, New York, NY 10010, U.S.A. (Received 3 March 1989 and accepted in revised form 5 April 1989) The transport rates of radiolabeled ascorbic acid (AA) and dehydroascorbic acid, as well as 3-0methyl-D-glucose and L-glucosefrom blood into aqueous humor, lens epithelium and lens ' cortex' compartments were studied in male Sprague-Dawley rats. In vivo pulse chase kinetic studies and modeling of transport from plasma and aqueous and on into idealized water compartments of lens epithelium and cortex allowed for the calculation of transport rate constants, K~ (min-1), in experiments utilizing L-glucose as a passive internal control. TLC chromatography was used to monitor intraocular labeled molecules deriving from labeled test molecules introduced via blood. Results indicate that AA enters aqueous humor at rates similar to L-glucose and likely via simple passive diffusion. In contrast, an active uptake of AA by lens epithelium was found with the calculated entry constant for aseorbate being more than 21 times faster than that of L-glucose. Concentrations in lens epithelium were found to be more than twice that of aqueous humor within only 7 miu from the introduction of a [x~C]AAbolus into blood. It was also found that very little AA continued on past the epithelium to the interior lens cortex compartment. Our data suggest no special uptake of AA by lens fiber cells. The non-metabolizable analog of D-glucose, 3-0methyl-n-glucose, however, readily moves past the lens epithelium into fiber cells at much faster rates than the passive L-glucose marker and in a manner consistent with facilitated diffusion. The data suggest that even in a nocturnal species, such as a rat, which demonstrates relatively low circulating levels of ascorbic acid in plasma and aqueous humor, special mechanisms exist for moving ascorbic acid into intraocular tissues. More specifically, the lens epithelium actively takes up ascorbate for some, as yet unclear purpose while the interior fiber cells appear to have no special uptake mechanism for this molecule. Key words: ascorbic acid; dehydroascorbic acid; aqueous humor; lens epithelium; lens; Vitamin C; transport; rat. 1. I n t r o d u c t i o n Most species such as r a t are able to g e n e r a t e ascorbic acid (AA) from D-glucose t h r o u g h gulonic acid, while others including guinea-pig, lower p r i m a t e s an d h u m a n s lack L-gulonolactone oxidase a n d rely on d i e t a r y sources of this v i t a m i n (Levine an d Morita, 1985; B u i - n g u y e n , 1985; Burns, 1975; Ch at t er j ee, M a j u m d e r , N a n d i a n d S u b r a m a n i a n , 1975). A l t h o u g h no clear p ict u r e of A A f u n c t i o n in t h e eye or m o r e specifically, lens, has evolved, m a n y studies h a v e n o t e d its p o t e n t i a l i m p o r t a n c e . A p o t e n t i a l role has been suggested for A A in m o d u l a t i n g lens m e t a b o l i s m v i a t h e hexose m o n o p h o s p h a t e s h u n t (Pirie, 1965; V a r m a , B a u e r an d Ri ch ar d s, 1987). A A has also been n o t e d to be an effective chemical cofactor in o x y g e n - d e p e n d e n t h y d r o x y l a t i o n reactions, a l t h o u g h its r e q u i r e m e n t is n o t specific (Stetten, 1949; Axelrod, U d e n f r i e n d a n d Brodie, 1954; S tau d i n g er , K r i s c h an d Leo n h au ser , 1961; Z a n n o n i and L a Du, 1960). O t h e r s u g g e s t ed f u n c t i o n s for A A in t h e eye include p o t e n t i a l roles as b o t h o x i d a n t , a n t i - o x i d a n t an d free radical s c a v e n g e r p r e v e n t i n g * This work has been supported by Research Grant EY 04418 from The National Institute of Health (NEI), Bethesda, MD, U.S.A. t Correspondence address. 0014-4835/89/110873 + 13 $03.00/0
9 1989 Academic Press Limited
874
J. DIMATT10
oxidative damage and thereby maintaining an intraocular homeostasis Varma, Ets and Richards, 1977; Bhuyan and Bhuyan, 1977; Delamere, Paterson and Cotton, 1983; Giblin, McCready, K o d a m a and Reddy, 1984; Delamere and Williams, 1985; (Riley, Schwartz and Peters, 1986). We have recently presented in vivo evidence suggesting t h a t ascorbate enters aqueous humor of guinea-pigs via an active transport process (DiMattio and Streitman, 1986, 1987) and t h a t AA is the preferred form over dehydroascorbic acid with regard to transport into the eye. In addition, TLC studies indicated t h a t even with a pure dehydroascorbic step pulse delivered into blood, only labeled AA was found in aqueous humor suggesting t h a t AA is the only transported species of physiological importance to the guinea-pig eye. In contrast, parallel studies with rat indicated t h a t AA movement into aqueous humor was primarily via simple passive diffusion, as was that of dehydroascorbic acid. Moreover, TLC radiolabeled tracer studies revealed t h a t only the reduced AA was found in aqueous humor, suggesting t h a t dehydroascorbic acid was converted to AA before or during its entry into aqueous humor. This suggested that reduced AA is the physiologically important species also in the rat. No synthesis of AA is known to occur in lens (Kuck, 1970) yet its significant presence in lens of m a n y species (Varma, 1987) suggests t h a t aqueous humor and ciliary process are a likely route of passage. Serious questions remain, however, as to how the ascorbate moiety moves from blood to lens in the various species. Recent in vitro studies with whole lens of guinea-pig, bovine and humans suggested that mediated transport of dehydroascorbic acid occurred while uptake of reduced ascorbate was found to be negligible (Kern and Zolot. 1987). This left unclear how the lens achieves its high reduced AA and low dehydroascorbate levels. Moreover, other studies had reported t h a t both AA and dehydroascorbic acid were taken up at similar rates by lenses of rat and guinea-pig, indicating no carrier mediation for either species (Hughes and Hurley, 1970) and t h a t dehydroascorbic acid readily penetrated rabbit lenses in vitro (Shimuzu, 1965; van Heyningen, 1970). I t is the purpose of this work to help clarify the mechanism whereby ascorbic acid is brought into the lens. This was done by following the in vivo movement of labeled test molecules of AA, dehydroascorbic acid, L-glucose and 3-O-methyl-D-glucose from blood to aqueous humor, and subsequently into lens epithelium and lens interior or ' c o r t e x ' water compartments. Our work indicates that in the normal albino rat, which can produce AA, little or no carrier mediation at the level of the iris-ciliary process or the blood-aqueous barrier is present, but clear carrier mediation at the level of the aqueous-lens epithelium involving an active energy-requiring mechanism was observed. In contrast, little ascorbic acid was found to enter into the interior cortex water c o m p a r t m e n t in our experiments leading us to conclude that uptake by lens fiber cells is slow at best. Our work suggests t h a t the lens epithelium m a y be an important locus of ascorbic acid function, and t h a t special mechanisms exist in the rat lens epithelium which can meet this requirement.
2. M a t e r i a l s a n d M e t h o d s Current methods extend and improve those introduced previously (DiMattio, 1984). In short, the experimental procedure consisted of the introduction of a double label bolus of [3H]L-glucose (L-glu) and either [14C]L-ascorbic acid (AA), dehydroascorbic acid (DHA) or [14C]3-O-methyl-o-glucose (mD-glu) at time zero into the circulation of an anesthetized
ASCORBIC ACID TRANSPORT INTO LENS E P I T H E L I U M
875
(Nembutal : 50 mg kg -1, i.p.) and cannulated test rat (male Sprague-Dawley ; 250-300 g) and determining the decay in concentration with plasma samples taken periodically. At the prescribed end of the experimental period~ during which labeled molecules moved freely from blood to intraocular tissues, the animal was killed and samples of aqueous humor (10-20/el) were obtained. At this time the lens was removed and dissected to separate the capsuleepithelium from the interior cortex. The epithelium and cortex samples were quickly weighed and set to dry (60~ ; 24 hr). The following day the dry samples were weighed and returned to the oven until no further change in dry weight was observed (usually 3 days). The dried samples were then dissolved in tissue solubilizer (0"5 N quaternary ammonium hydroxide in toluene; Beckman BTS 450) and isotopic concentrations of labeled test species were determined (cpm/mg dry weight) via liquid scintillation spectrometry (Beckman LS4000) using Dimiscent scintillation cocktail (National Diagnostics). Water content for each of the samples was determined and the isotopic concentration expressed as cpm m1-1 of tissue water using appropriate values for tissue water content. The blood samples were centrifuged (600 g, 10 min) and 50/el samples of plasma were counted using Liquiscent scintillation cocktail (National Diagnostics). Double channel counting for 14C- and 3H-labels was used and the results were computer processed to correct for quenching and channel spillover and uniformly reported as dpm m1-1. The plasma data were fitted to a double exponential decay function with time. t (rain) from 0 to T min, when the animal was killed with an overdoes of sodium pentobarbital. Death was defined as the complete cessation of monitored cardiac activity and usually took less than 15 sec after lethal pentobarbital administration. This plasma decay function along with aqueous, lens epithelium and cortex concentration data was used to calculate transport rate constants which give a measure of how fast the concentrations of test molecules in a specific compartment change with time. Calculation of rate constants The plasma isotopic concentration data Cp(t) vs. time, t, for each test molecule were fitted graphically to a double exponential decay curve of the form : Cp = A + Be -bit + Ce-b2 t,
(1)
where A,B,C,b~ and be are determined constants. Constant A has been shown (DiMattio and Zadunaisky, 1981) to be the plasma concentration at t = oc, Cp(oc). Each experiment curve was normalized by dividing by a constant Cp(0), the plasma concentration at the onset of the experiment. The constants were determined graphically and via curve fitting using Asystant software (Macmillan Software Co., N.Y.). This program provides 'goodness of fit parameters' and resulted in plasma constants with a correlation coefficient of 0"992 or better. Thus, each experiment resulted in a specific plasma curve, with well-known defined constants, which were subsequently used to calculate appearance curves and entry rate constants for aqueous and lens compartments. Figure 1 illustrates the transport scheme used to derive appearance curves. For transport from blood plasma to aqueous humor, a simplified linear first-order system equation was used : dCA/dt = K~,Cp-KAo C,~. (2) T h i s equation indicates that the rate of change of concentration in aqueous humor is dependent simply on linear entry and exit rates with defined rate constants, KA~ and KAo. Note at t = oo. KA,/KAo = CA/Ca (oc). (3) For simple diffusion and carrier facilitated diffusion, the two adjacent compartments (e.g. plasma-aqueous) ideally come to a transport steady state with concentrations nearly equal and thus, CA/Cp(~) = 1"0 and Ka, = KAo = KA.
(4)
Another possibility is that C A / C e ( ~ ) > l'0 suggesting that some mechanism such as an active transport carrier is present which allows the accumulation of label to concentrations higher than those from which it originated.
876
J. D I M A T T I O
{ P)
Aqueous (A)
KA,
_
d~ ";';- : K,I C p - ~ , CA
IAqueous (AI
•/
Lens
- ~ : ~,C, -KEoC~
K,o
~-r : Kc, cr-KcoCc
FIo. 1. Transport schemes : from plasma (P) to aqueous (A) and from aqueous to lens epithelium (E) and lens interior 'cortex' compartments. Subscripts for rate constants, K (min -1) refer to destination compartment such as aqueous (A), epithelium (E) or cortex (C) with i refering to entry (in) and o, to exit (out) notations. Concentration, C, is dpm m1-1 and t, time (min-). W e limit o u r analysis to simple, carrier-facilitated diffusion a n d a c t i v e t r a n s p o r t m e c h a n i s m s for t h e t e s t molecules studied. U s i n g L-glucose as a control or passive m a r k e r allowed us to c o m p a r e results w i t h t h e o t h e r t e s t molecules a t least o n some r e l a t i v e basis in t h e same e x p e r i m e n t a l animal. As s h o w n previously, ( D i M a t t i o a n d Z a d u n a i s k y , 1981; D i M a t t i o , 1984), t h e a q u e o u s h u m o r c o n c e n t r a t i o n versus t i m e a p p e a r a n c e f u n c t i o n derived from e q n (1), a n d e q n (2) was s h o w n to be of t h e f o r m :
where
CA(t) = Sl(Anl + AA2 e-~n~ +An a e-b~' +An4 e-O~'),
(5)
S 1 - Cn ( ~ ) -- KA~
(6)
Cp(~) and
Kno
Anl = CA ( ~ ) / S~ = C e ( ~ ) = A
KnoB
KAoC
bl-KAo
b2-Kno
A n 2 = - - ~ - -
Knob
Ana - - -
Kno--b~
KnoC An4 = KAo_b2.
A
(7) (8)
(9)
(10)
A c o m p u t e r trial a n d error solution to eqns (4-6) allows for t h e e s t i m a t i o n of t r a n s p o r t c o n s t a n t s KA, a n d Kno using t h e d e t e r m i n e d p l a s m a c u r v e c o n s t a n t s of e q n (1), explicit a q u e o u s c o n c e n t r a t i o n d a t a a t a specific time, T, a n d some e s t i m a t e of t h e t r a n s p o r t s t e a d y state.
A S C O R B I C ACID T R A N S P O R T I N T O L E N S E P I T H E L I U M
877
Since lens epithelium is taken to be b a t h e d b y aqueous h u m o r and the concentration of labeled molecule in aqueous is represented by eqn (4), this equation is then used as the forcing function for transport into lens epithelium. Thus, the known constants of eqn (4), along with lens epithelium concentration d a t a at time, T, and some estimate of lens e p i t h e l i u m / a q u e o u s steady state are then used to determine the lens epithelium e n t r y and exit rate constants, Kz, and KEo, respectively. Transport rates into lens epithelium were taken as simple first order as was done for aqueous formation. The system equation is:
d C J d t = Ks~CA-Kso C~.
(11)
where E refers to ' Epithelium '. As a first approximation, the concentration of test molecule was assumed to be uniform t h r o u g h o u t the lens epithelium water c o m p a r t m e n t , with no distinction being made between intracellular or extracellular water. The equation t h a t describes the appearance and disappearance of labeled test molecule in lens epithelium with time is : C~(t) = 82(B~1 + BB~ e-KE~ + B.3 e-KA~ + B ~ e-blt + BB5 e-~2t),
(12)
S 2 - CA(~176 Kso
(13)
B m = C~ (oo)/S 2 = CA ( ~ )
(14)
where and
B,~-
AAd%~ AA3K~~ AAaK~~ KAo_K~~ +bl-~-~oo-+ b2-K~o AA,
(15)
AAd~.o -
(16)
B,3 = -
K.o-KA.
BB4 B~, -
AA~K.o
(17)
KEo-b~ AA4Kso
(18)
K~o-b2
Since lens cortex or interior tissue c o m p a r t m e n t is largely surrounded by epithelium we assume t h a t tracer molecules enter only from across the epithelium. Thus, in a parallel m a n n e r we used the concentration of test molecules as described in eqn (12) to be the forcing function for transport into a defined interior lens water c o m p a r t m e n t taken as 'cortex ', C. Again, transport into lens cortex is considered arising from simple first order rate expressions and the system equation is: dCc/dt = Kc, C~ -Keo Cc.
(19)
Using eqn (12) along with the known coefficients and constants, Laplace analysis was again used to derive an appearance and disappearance function ibr the ' c o r t e x ' or interior fiber cell c o m p a r t m e n t concentration, Cc(t ), given below: Cc(t ) = S3(Cc~+Cc~e-KC~176 where and
ga~
(20)
S 3 - Cc (oo) _ K~,
(21)
Cot = Cc ( ~ )/ S 3 = CE (oc)
(22)
C.(~)
K~o
Ce2 = B":'I(c~ + BsaKc~ .----_-7-B'4Ke~ BBsKc~ Keo-Kco KAo-Kco I-b~--Kco q b2-Kco Cca =
B.d;~o
eel
(23)
(24)
878
J. DIMATTIO cc4 - -
B.~co -
K~o-K~o
Cc~ -
B.4Kco -
-
K~o-bl
(25)
(26)
B.~K~o Cc~ -
Kco_b2.
(27)
Implied in our analysis are common simplifying assumptions concerning mixing and preservation of label on the molecule of interest. We note that small compartment volumes and convective circulation in aqueous humor, coupled with diffusion of small molecules, assists in mixing components of aqueous humor. I t is further noted that since nearly all the aqueous and complete lens tissue samples were taken, we are assured that at least uniform samples and average concentrations were used for rate constant calculations. Radio thin-layer chromatographic techniques were used to monitor the radiolabeled molecules in aqueous and tissue samples. Appropriate standards and labels were run on TLC plates and scanned with a Radiomatic Radioscanner RS with RTLC analysis software. Labeled L-glucose and 3-O-methyl-D-glucose were run on G-plates (Analtech, Newark, Delaware) using benzene : methanol : acetone : acetic acid (70 : 20 : 5 : 5) and ran with l~f's of 0-178 and 0"272, respectively, using methods outlined previously (DiMattio, 1988). Since ascorbic acid and dehydroascorbic acid are unstable in air a n d / o r water, all samples were spotted and analyzed under 100% nitrogen and confirmed using glass TLC silica gel H plates, custom prepared by Analtech with 6 % metaphosphoric acid to stabilize the ascorbic acid and prevent oxidation during analysis. The metaphosphoric acid H plates were run with acetonitrile :butyronitrile :water (66:33:2) and gave Rf's of 0-16 and 0"28 for ascorbic acid and dehydroascorbic, respectively.
3. R e s u l t s W a t e r c o n t e n t s of normal, male S p r a g u e - D a w l e y r a t s lens e p i t h e l i u m a n d lens ' c o r t e x ' c o m p a r t m e n t s were d e t e r m i n e d as functions of b o d y weight a n d age. W e n o t e t h a t for t r a n s p o r t e x p e r i m e n t s , r a t s used r a n g e d from 245 to 335 g, w i t h a m e a n of 296+_28 g. W e f o u n d t h a t lens e p i t h e l i u m % w a t e r c o n t e n t did n o t v a r y a p p r e c i a b l y w i t h r a t size a n d was t a k e n as 79"2 +_0"8 (n = 18) for calculations. Lens cortex w a t e r c o n t e n t was f o u n d to decrease w i t h age a n d weight. W e f o u n d 250 g r a t s h a d a lens cortex w a t e r c o n t e n t of 57-1 ___0"4 % (n = 9), 350 g rats, 55" 1 +- 0"3 % (n = 6), a n d 450 g rats, 54"2_ 0"3 % (n = 5). A p p r o p r i a t e values were used to calculate lens tissues c o n c e n t r a t i o n s in d p m m1-1 water. F i g u r e 2 illustrates compiled curves n o r m a l i z e d to the p l a s m a c o n c e n t r a t i o n a t t = 0, (Cpo), of c o n c e n t r a t i o n C vs. t (min). I t is n o t e w o r t h y t h a t A A leaves t h e p l a s m a f a s t e s t i n d i c a t i n g t h a t t h e b o d y r e m o v e s ascorbic acid efficiently from blood p r o b a b l y in an effort a t conservation. T a b l e I r e p o r t s m e a n curve fitting c o n s t a n t s of eqn (1) to i n d i v i d u a l curves o b t a i n e d in e x p e r i m e n t s . F i g u r e 3 r e p o r t s our c o m p i l e d results for a p p e a r a n c e of t h e v a r i o u s labeled molecules in aqueous h u m o r . N o t e t h a t [14C]3-O-methyl-D-glucose moves into aqueous h u m o r f a s t e s t of all t h e molecules t e s t e d a n d t h a t this n o n - m e t a b o l i z a b l e t e s t analog of D-glucose was p r e v i o u s l y shown to e n t e r b o t h aqueous a n d v i t r e o u s h u m o r in m a n n e r consistent w i t h f a c i l i t a t e d diffusion. These d a t a are consistent w i t h our p r e v i o u s r e p o r t s for whole lens (DiMattio, 1984). T a b l e I I r e p o r t s our results for p l a s m a to aqueous e n t r y r a t e c o n s t a n t calculations for the v a r i o u s molecules studied. N o t e t h a t we r e p o r t calculations based on 7 min a n d 13 min e x p e r i m e n t s . This was
ASCORBIC ACID T R A N S P O R T INTO LENS E P I T H E L I U M
879
1-000 o .... o ~ ~ =--= Q-- =
0.900 0.800
L-glucose L-Ascorbic acid 5 - O - m e t h y t - glucose dehydrocscorbic acid
0"700
g 0.600 "--~ 0.500
=
o.
LLL"
oo
0.2oo
J " ~ ; ~ ~ ~ - l ~
o.loo
~
.
~
~
0.000 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
IO
_
_
-1-
~
,. . . . . . . . . . . . . . . . . . . . 4.0 50
50
20
~
60
Time (rain)
Fie. 2. Plasma concentration vs. time (min) curves resulting from a bolus of labeled test molecular species introduced into a central plasma compartment at time 0. The concentrations are normalized to the concentration at time 0 or Cpo. Each curve represents a compilation from at least 12 experiments. TABLE [
Mean curve constants derived from a double exponential fit of plasma concentration vs. time (rain) data n*
A
lUll]L-glucose
(15)
0
[z4C]L-Ascorbic acid
(12)
0
[t4C]Dehydroascrobic acid
(7)
0
[14C]3-0-Methyl-D-glucose
(12)
0
B
C
0"383 0"617 +0"018 __+0'018 0"292 0"708 +0"077 • 0"366 0-634 -+0"073 -+0-073 0'456 0"544 -+0"008 -+0-008
b1
b~
0"0278 +0"0031 0'0475 -+0"0146 0"0409 -+0-0067 0"0195 __+0'0079
0"311 +0"039 0"488 +0"113 0"597 -+0'139 0"550 4-0'184
* n equals the number of experiments. Constants A, B, C, bl, b2 are defined by eqn (1) and are reported with S.D. below.
done to help s u p p o r t t h e n o t i o n t h a t t he m o d e l i n g of t r a n s p o r t was at least reasonable. We r e p o r t t h a t p l a s m a - a q u e o u s e n t r y r a tes for ascorbic acid were n o t found to be significantly different from those of L-glucose. This, along with ascorbic acid s a t u r a t i o n e x p e r i m e n t s with cold L-ascorbic acid an d t h e lack of a n y effect of glucose t r a n s p o r t inhibitors p h l o r e t i n (1 mM) a n d phloridzin (1 mM) on ascorbic acid m o v e m e n t , allowed us to suggest t h a t ascorbic acid e n t r y into aq u eo u s h u m o r of t h e r a t occurs p r i m a r i l y via simple passive diffusion (DiMattio, 1988). I n addition, we d e m o n s t r a t e d t h a t ascorbic acid enters a q u e o u s h u m o r unchanged, r e m a i n i n g in t h e reduced form, while d e h y d r o a s c o r b i c acid en t er ed or at least was f o u n d in a q u e o u s
880
J, DIMATTIO Rat
1'200 blOC 1,000 0'90C
I,
0,80C
o.7oc o.6ooi 0.500
i
s
0.400 0'500 0.200 0,100 0.000
.
0
.
.
I
i
.
IO
L-glucose ~ - - o Ascorbic acid , - - , 5-O-methyl-O-glucose A - - , Dehydro(:]scorbic acid " - - " .
.
I
20
.
.
.
.
I
50
.
.
.
.
l
40
.
.
.
.
l
.
.
.
50
.
I
60
.
.
.
.
I
.
.
70
.
.
l
.
.
i
,
80
I
90
Time (min)
Fro. 3. Illustrates the appearance of radiolabeled test molecules into aqueous. It is reported as the concentration in aqueous divided by the plasma concentration as a fucntion of tinie (min). TABLE I I
P l a s m a to aqueous humor entry rate constants, KA~
[all]L-glucose [14C]L-Ascorbicacid [14C]Dehydroascorbic acid [14C]3-O-MethylD-glucose
Ca/CP KA~ (1 per min)
CA/CP KAi (1 per min)
7-0 min
13"0 min
0"202+_0'021 0.0220+_@0018 0"352+_0"041 (9) (9) (13) 0-0286_+0.046 0"0236+,0"0029 0-561• 0'057 (5) (5) (8) 0-354_ 0"059 0"0357_+0.0049 0-693+ 0"07l (5) (5) (6) 0"671 +_0"071 0'102_+0"008 0"889_+0"059 (5) (5) (9)
0'0213+,0"0030 (13) 0"0243_+0"0038 (8) 0'0394 + 0"0058 (6) 0"0977+_0"006 (9)
Transport S.S. 1"0 1"0 !'0 1'0
o n l y in t h e r e d u c e d or ascorbic acid form (DiMattio, 1988). Thus, t h e fact t h a t d e h y d r o a s c o r b i c acid e n t r y r a t e s are a b o u t 40 % higher t h a n for ascorbie acid could reflect differences in passive p e r m e a b i l i t y p r o p e r t i e s between t h e two molecules or a m i n o r c o n t r i b u t i o n via carrier m e d i a t i o n . I n a n y event, 3-O-methyl-D-glucose e n t r y r a t e s into aqueous h u m o r were f o u n d to be b y far the f a s t e s t while L-glucose, ascorbic acid a n d d e h y d r o a s c o r b i c acid e n t e r e d into a q u e o u s h u m o r a t c o m p a r a b l y low r a t e s a n d p r i m a r i l y v i a passive diffusion. F i g u r e 4 r e p o r t s our c o m p i l e d e x p e r i m e n t a l results of t h e r a t i o of c o n c e n t r a t i o n in lens e p i t h e l i u m w a t e r c o m p a r t m e n t to t h a t f o u n d in aqueous a t v a r i o u s e x p e r i m e n t end times. Since e p i t h e l i u m to a q u e o u s h u m o r r a t i o s m u c h g r e a t e r t h a n 1"0 were seen in r e l a t i v e l y s h o r t t i m e periods, more t h a n simple or f a c i l i t a t e d diffusion is required t o e x p l a i n these results. I n c o n t r a s t , L-glucose a n d 3-O-methyl-D-glucose a p p r o a c h similar e q u i l i b r i u m c o n c e n t r a t i o n s r e l a t i v e to t h e circulating plasma. Also note t h a t
A S C O R B I C ACID T R A N S P O R T INTO L E N S E P I T H E L I U M
881
6,000 5.500
Tsl
5.000 4.500
.-:4 . 0 0 0 -~ E _= =la -~
5.500
/
5.000
xL
o ~ o -z~--
o L- glucose o ,Ascorbic acid z~ 3 - O - m e t h y t - D - g l u c o s e
2.500
f / / ~ ~j"~i~i0
~
0.000",~
~
0
,,, I0
~ _ ~ , 20
i,
~_,
50
.... 40
J .... 50
~ ..... 60
,,, 70
~ j ~ _ ~ 80
90
Time (rain)
FIG. 4. Illustrates the appearance of radiolabeled test molecules in the lens epithelium compartment. It is reported as epithelium concentration divided by the aqueous concentration. The number of experiments for each point is at least five while the n for the 7 and 13 min points are given in the tables.
the n-glucose analog reaches this equilibrium concentration significantly faster than the L-glucose, indicating a facilitating factor involved in transport. Figure 4 also illustrates t h a t our experimental results with ascorbic acid are significantly different and much faster. Concentration ratios as high as 5-0 were observed, indicating that an overall epithelium water c o m p a r t m e n t ascorbic acid concentration five times that of aqueous humor occurred within 20 min after introduction of the label bolus into blood. Thus, some mechanism is present which causes ascorbic acid to be accumulated in lens epithelium. This mechanism must be energy-requiring since aseorbic acid must be moved against a concentration gradient to achieve the concentrations noted by us. These factors suggest an active transport mechanism. Table I I I reports our rate constant calculations using 7- and 13-min experiments. T A ~ E III
Aqueous humor to lens epithelium entry rate constants, KE~
[all]L-glucose [I~C]L-Aseorbie acid [14C]3-O-MethylD-glucose
C J C a Ke, (1 per min)
C J C a Ks~ (1 per rain)
7'0 min
13"0 min
Transport S.S.
0-253+0-047 0.0283+__0"0031 0"411--+0"031 0'0261-+0'0028 (9) (9) (13) (13) 2-06 • 0-14 0-617-t-0"047 3"42_+0"21 0"672+0-054 (5) (5) (8) (8) 0.571 -+0-050 0-198__+0"015 0.803-+0-018 0.194+0-014 (5) (5) (9) (9)
1"0 5"0 1.0
* Values are listed as means _+s.D. with the number of experiments in parentheses below. 3O
E E R 49
882
J. DIMATTIO
Our calculated rate c o n s t a n t s indicate t h a t ascorbic acid e n t r y rates are more t h a n 20 times faster t h a n L-glucose, which was considered to be a comparable m a r k e r in size a n d molecular characteristics. These results indicate an active u p t a k e m e c h a n i s m for ascorbic acid b y lens epithelium. We note t h a t rate c o n s t a n t calculations utilized a n estimated t r a n s p o r t s t e a d y - s t a t e value reported in our results. A n y error in this e s t i m a t e would only m o d e r a t e l y effect our rate c o n s t a n t estimate, b u t would n o t alter results t h a t depend on actual d e t e r m i n e d c o n c e n t r a t i o n e n c o u n t e r e d a t specific times after the bolus i n t r o d u c t i o n .
IqO0
~
I.ooo
0.900
i
~
~
•
~ • T A
0-800
T/L
j o7oo
0.600
/I
e
L-glucose o - - o
0.2 0~ /
T/
AsCOrblcacid e - - e 5-0- rnethy[- D-glucose ~ - - a r
o
0
10
20
50
40 50 Time (rnin)
60
,
,
L
,
~
70
~
,
,
i
I
80
i
i
i
I
90
FIG. 5. Illustrates our data on the appearance ofradiolabeled molecules in the 'cortex' compartment. It is reported as cortex concentration divided by epithelium concentration. The number of experiments for each point is at least five. TABLE IV
Lens epithelium to cortex entry rate constants, Kc~
[all]L-glucose [x4C]L-Ascorbic acid [14C]3-O-Methyl])-glucose
Cc/C~ Ke, (1 per min)
Cc/CE Kci (1 per rain)
7"0 min
13"0 rain
Transport S.S.
0'092_+0"034 0"0101__0"0031 0"166_+0'053 0"0108__+0'036 (9) (9) (13) (13) 0'009_+0"0030"00178_+0"00041 0"020_+0"006 0"00167_+0-00048 (5) (5) (S) (S) 0"388_+0-049 0"175_+0-016 0"642_+0"084 0"180_+0"018 (5) (5) (9) (9)
1-0 1'0 1'0
* Values are listed as means-+s.D, with the number of experiments in parentheses below. Figure 5 reports our results with the lens interior or ' c o r t e x ' c o m p a r t m e n t . While ascorbic acid m o v e m e n t into lens e p i t h e l i u m was very fast, m o v e m e n t into the interior lens c o m p a r t m e n t was seen to be very slow. R a t e c o n s t a n t s calculated in p a r t
ASCORBIC ACID TRANSPORT INTO LENS EPITHELIUM
883
from the data of Fig. 5 are reported in Table IV and show in fact that entry into a mean interior lens water c o m p a r t m e n t is significantly slower even than that of Lglucose. Thus, if we assume L-glucose movement into lens cortex is via passive diffusion as the data suggest, then no carrier appears to be involved in moving ascorbic acid. In contrast, our data indicate t h a t the D-glucose analog enters lens quickly and efficiently at rates more than ten times those of L-glucose. Moreover, our results with mn-glu suggest that a facilitated transport mechanism is involved in glucose uptake by lens fiber cells since concentration ratios do not rise above 1.0. Thus, glucose transport into lens ' cortex' or interior fiber cells, is fast, but does not proceed against a concentration gradient ruling out active transport. 4. D i s c u s s i o n In a recent review on aseorbic acid and the eye, Varma has suggested that aqueous humor could act as a source for ascorbate for lens and cornea in species such as man, ox and rabbit which have high levels of ascorbate in aqueous humor (Varma, 1987). The rat, however, did not appear to fit in with this theory since little ascorbate was found in either plasma or aqueous humor. Recent work in this laboratory (DiMattio, 1986 ; 1988) with normal rats has revealed t h a t although low plasma ascorbate levels of 3"3+_0-8 mg per 100 ml (n -- 16) were detected, we have also found t h a t labeled aseorbic acid (MW 176) enters into aqueous humor at similar rates as the passive marker L-glucose (MW 180}, and t h a t the rate of entry into aqueous was not saturable with unlabeled ascorbate nor affected by the drugs phloretin (10 -3 M) and phloridzin (10 -3 M). These and other (Delamere and Williams, 1987) observations led us to conclude that ascorbic acid entry into aqueous humor was primarily via simple passive diffusion. Thus, aqueous humor ascorbate levels should be relatively low, reflecting low plasma levels. In fact, we found ascorbate levels to be even lower than in plasma, 2 " l + 0 . 5 m g per 100ml (n = 10), or about 60-70% of the circulating plasma level. This low ascorbate concentration in aqueous humor could well arise from uptake or utilization of ascorbate by intraocular tissue. In this report we found t h a t ascorbate is taken up by lens epithelium even against a concentration gradient. This observation could explain the low ascorbate levels found in aqueous by pointing to where at least some of the ascorbate had gone. A major finding in this work is t h a t while lens epithelium actively took up ascorbate, lens interior fiber cells in our ' c o r t e x ' compartment, apparently, did not. Lens fiber cells were seen to take up quickly and efficiently the non-metabolizable glucose analog [14C]3-O-methyl-D-glucose in a manner consistent with facilitated diffusion, but demonstrated little ability to take up ascorbate. Uptake rates for aseorbate were found to be significantly lower than even L-glucose. I t must be pointed out, however, t h a t we treat the whole lens interior as a single uniform c o m p a r t m e n t using average values, which m a y misrepresent the actual case and mask higher uptake levels by some regional fiber cells. We do not believe this to be the case, however, since L-glucose presumably moves into this interior c o m p a r t m e n t via passive diffusion and ascorbate should at least follow this pattern. We therefore suggest t h a t while ascorbate is actively taken up by lens epithelium, interior lens fiber cells demonstrate no interest in this molecule. This m a y be very important in attempting to understand where and what m a y be the essential functions of ascorbic acid which to date have remained somewhat elusive. In addition, the presence of capsule in our 'epithelium' c o m p a r t m e n t could also reflect a role for AA in capsule collagen formation. 30-2
884
J. DIMATTIO
These findings appear to support the notion t h a t lens epithelium and fiber cell are not metabolically coupled. In addition, the facilitated D-glucose transporting system does not appear to facilitate ascorbic acid uptake as has been suggested to occur in other systems (Khatami, Li and Rockey, 1986). This is further complicated by other reports which suggest t h a t D-glucose and dehydroascorbic acid may share a common carrier (Bigley, Wirth, Layman, Riddle and Stankova, 1983). These issues remain unclear. Our in vivo system is complicated by geometry and complex intercellular relationships. I t m a y be that lens epithelium, by taking up ascorbate, does not allow it to proceed further to interior fiber cells to any great extent, while D-glucose more readily crosses this epithelium. Moreover, lens fiber cell D-glucose carriers, like neutrophils and fibroblasts, m a y require the dehydroascorbate moiety and do not facilitate AA movement into cells significantly. Hughes and Hurley (1970) have reported t h a t both ascorbic acid and dehydroascorbic acid were taken up by the lens of rat and guinea-pig at similar rates. We note, however, t h a t lenses had been previously isolated and stored in saline and, most importantly, no a t t e m p t was made to distinguish between epithelium and cortical uptake. In fact, no previous study of our knowledge has made this distinction. Without making any clear identification of differences in uptake between lens epithelium and interior cortical fiber cells, results could easily mask high or low uptake by these tissues. A more recent study with calf lens has suggested that only dehydroascorbic acid enters whole calf lens in a manner which appears to be related to facilitated glucose transport (Kern and Zolot, 1987). However, in this same study reported values of ascorbic acid and dehydroascorbic acid found in aqueous humor and lens indicate clearly that ascorbic acid has a significant presence in both tissue compartments while dehydroascorbic acid is nearly absent and amounts to only 3 - 1 0 % of the ascorbie acid concentrations. No direct comparison could be made, therefore, with this present work. I t has been previously noted that the presence of ascorbate in ocular tissue points to some as yet unknown function for this molecule. Our work further suggests t h a t even in a species which did not appear to have high ascorbate concentrations in ocular tissues, special mechanisms exist which bring this molecule into lens and more specifically into lens epithelium and likely for some purpose. ACKNOWLEDGMENTS The author is indebted to Mr Jack Streitman for expert technical assistance. The computer assistance of Mr Al Drayton is gratefully acknowledged. In addition, the author wishes to thank Dr V. Fisher for V.A. Hospital facilities without which this work would not have been possible. This work has been supported by Research Grant EY 04418 from the National Institute of Health (NEI), Bethesda, MD. REFERENCES Axelrod, J., Udenfriend, S. and Brodie, B. B. (1951). Ascorbic acid in aromatic hydroxylation. III. Effect of ascorbic acid on the hydroxylation of acetanilide, aniline and antipyrine in vivo. J. Pharmacol. Exp. Ther. l l l , 176-81. Bigley, R., Wirth, M., Layman, D., Riddle, M. and Stankova, L. (1983). Interaction between glucose and dehydroascorbate transport in human neutrofils and fibroblasts. Diabetes 32, 545-8. Bhuyan, K. C. and Bhuyan, D. K. {1977). Regulation of hydrogen peroxide in eye humors.
ASCORBIC
ACID
TRANSPORT
INTO
LENS
EPITHELIUM
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Effect of 3-amino-lH-1,2,4-triazole on catalase and glutathione peroxidases of rabbit eye. Biochim. Biophys. Acta 497, 641 51. Bui-nguyen, M. H. (1985). Ascorbic acid and related compounds. Chromatographic Sci. 30, 267-301. Burns, J. J. (1975). Overview of ascorbic acid metabolism.. Ann. N.Y. Acad. Sci. 258, 5-7. Chattergee, I. B., Majnmder, A. K., Nandi, B. K. and Subramanian, N. (1975). Synthesis and some major functions of vitamin C in animals. Ann. N.Y. Acad. Sci. 258, 24-47. Delamere, N.A., Paterson, C.A. and Cotton, T . R . (1983). Lens cation transport and permeability changes following exposure to hydrogen peroxide. Exp. Eye Res. 37, 45-53. Delamere, N. A. and Williams, R . N . (1985). Detoxification of hydrogen peroxide by the rabbit iris-ciliary body. Exp. Eye Res. 40, 805-11. Delamere, N.A. and Williams, R . N . (1987). Comparative aspects of ascorbate in the ~queous humor. Invest. Ophthalmol. Vis. Sci. 28 (Suppl. 3), 74. DiMattio, J. A comparative study of ascorbic acid entry into aqueous and vitreous humors of the rat and guinea-pig. Invest Ophthalmol. Vis. Sci. (in press). DiMattio, J. and Streitman, J. (1986). In vivo ascorbic acid transport across ocular barriers and into retina of the normal rat. Invest: Ophthalmol. Vis. Sci. 27 (Suppl. 3), 350. DiMattio, J. and Streitman, J. (1987). A comparison of vitamin C and glucose transport across ocular barriers of rats and guipea-pigs. Invest. Ophthalmol. Vis. Sei. 28 (Suppl. 3), 74. Giblin, F. J., McCready, J. P., Kodama, T. and Reddy, V. N. (1984). A direct correlation between the levels of ascorbic acid and HeO 2 in aqueous humor. Exp. EyeRes. 38, 87 93. Hughes, R. E. and Hurley, R. J. (1970). In vitro uptake of ascorbic acid by the guinea-pig lens. Exp. Eye Res. 9, 175-80. Kern, H. L. and Zolot, S. L. (1987). Transport of vitamin C in the lens. Curt. Eye Res. 6, 885-96. Kh~tami, M., Li, W. and Roekey, J. H. (1986). Kinetics of ascorbate transport by cultured retinal capillary pericytes. Invest. Ophthalmol. Vis. Sci. 27, 1665-71. Kuck, J. F. g. (1970). Chemical constituents of the lens. In Biochemistry of the Eye. (Ed. Graymore, C. N.). Pp. 184 260. London: Academic Press. Levine, M. and Morita, K. (1985). Ascorbic acid in endocrine systems. Vit. Horm. 42, 2-64. Pirie, A. (1965). Glutathione peroxidase in lens and a source of hydrogen peroxide in aqueous humor. Biochem. J. 96, 244 53. Riley, M. V., Schwartz, C. A. and Peters, M. I. (1986). Interactions of ascorbate and H202 : implications for in vitro studies of lens and cornea. Curt. Eye Res. 5,207-16. Shimuzu, H. (1965). Experimental studies on the intake and the distribution of ascorbic acid in various tissues of the eye. Jpn. J. Ophthalmol. 9, 68-79. Staudinger, H. J., Krisch, K. and Leonhauser, S. (1961). Role of ascorbic acid in microsomal electron transport and the possible relationship to hydroxylation reactions. Ann. N.Y. Acad. Sci. 92, 195~207. Stetton, M. R. (1949). Some aspects of the metabolism of hydroxyproline studied with the aid of isotopic nitrogen. J. Biol. Chem. 181, 31 7. Van Heyningen, R. (1970). Ascorbic acid in the lens of the naphthalene-fed rabbit. Exp. Eye Res. 9, 38-48. Varma, S. D. (1987). Ascorbic acid and the eye with special reference to the lens. (Third Conference on Vitamin C.) Ann. N.Y. Acad. Sci. 498, 280-307. Varma, S. D., Bauer, S. A. and Richards, R. D. (1987). Hexose monophosphate shunt in rat lens: stimulation by vitamin C. Invest. Ophthalmol. Vis. Sci. 28, 1164-9. Varma, S. D., Ets, T. K. and Richards, R. D. (1977). Protection against superoxide radicals in rat lens. Ophthalmic Res. 9, 421-31. Zannoni, V.G. and La Du, B.N. (1960). Tyrosylurea resulting from inhibition of phydroxyphenylpyruvic acid oxidase in vitamin C-deficient guinea-pigs. J. Biol. Chem. 235(3), 2667-71.
Active Transport of Ascorbic Acid into Lens E p i t h e l i u m of the Rat* J O S E P t t DIMATTIO
Department of Physiology and Biophysics, New York University School of Medicine, 550 First Avenue, New York, NY 10016, U.S.A. and New York Veterans Administration Hospitalt, 408 First Avenue, New York, NY 10010, U.S.A. (Received 3 March 1989 and accepted in revised form 5 April 1989) The transport rates of radiolabeled ascorbic acid (AA) and dehydroascorbic acid, as well as 3-0methyl-D-glucose and L-glucosefrom blood into aqueous humor, lens epithelium and lens ' cortex' compartments were studied in male Sprague-Dawley rats. In vivo pulse chase kinetic studies and modeling of transport from plasma and aqueous and on into idealized water compartments of lens epithelium and cortex allowed for the calculation of transport rate constants, K~ (min-1), in experiments utilizing L-glucose as a passive internal control. TLC chromatography was used to monitor intraocular labeled molecules deriving from labeled test molecules introduced via blood. Results indicate that AA enters aqueous humor at rates similar to L-glucose and likely via simple passive diffusion. In contrast, an active uptake of AA by lens epithelium was found with the calculated entry constant for aseorbate being more than 21 times faster than that of L-glucose. Concentrations in lens epithelium were found to be more than twice that of aqueous humor within only 7 miu from the introduction of a [x~C]AAbolus into blood. It was also found that very little AA continued on past the epithelium to the interior lens cortex compartment. Our data suggest no special uptake of AA by lens fiber cells. The non-metabolizable analog of D-glucose, 3-0methyl-n-glucose, however, readily moves past the lens epithelium into fiber cells at much faster rates than the passive L-glucose marker and in a manner consistent with facilitated diffusion. The data suggest that even in a nocturnal species, such as a rat, which demonstrates relatively low circulating levels of ascorbic acid in plasma and aqueous humor, special mechanisms exist for moving ascorbic acid into intraocular tissues. More specifically, the lens epithelium actively takes up ascorbate for some, as yet unclear purpose while the interior fiber cells appear to have no special uptake mechanism for this molecule. Key words: ascorbic acid; dehydroascorbic acid; aqueous humor; lens epithelium; lens; Vitamin C; transport; rat. 1. I n t r o d u c t i o n Most species such as r a t are able to g e n e r a t e ascorbic acid (AA) from D-glucose t h r o u g h gulonic acid, while others including guinea-pig, lower p r i m a t e s an d h u m a n s lack L-gulonolactone oxidase a n d rely on d i e t a r y sources of this v i t a m i n (Levine an d Morita, 1985; B u i - n g u y e n , 1985; Burns, 1975; Ch at t er j ee, M a j u m d e r , N a n d i a n d S u b r a m a n i a n , 1975). A l t h o u g h no clear p ict u r e of A A f u n c t i o n in t h e eye or m o r e specifically, lens, has evolved, m a n y studies h a v e n o t e d its p o t e n t i a l i m p o r t a n c e . A p o t e n t i a l role has been suggested for A A in m o d u l a t i n g lens m e t a b o l i s m v i a t h e hexose m o n o p h o s p h a t e s h u n t (Pirie, 1965; V a r m a , B a u e r an d Ri ch ar d s, 1987). A A has also been n o t e d to be an effective chemical cofactor in o x y g e n - d e p e n d e n t h y d r o x y l a t i o n reactions, a l t h o u g h its r e q u i r e m e n t is n o t specific (Stetten, 1949; Axelrod, U d e n f r i e n d a n d Brodie, 1954; S tau d i n g er , K r i s c h an d Leo n h au ser , 1961; Z a n n o n i and L a Du, 1960). O t h e r s u g g e s t ed f u n c t i o n s for A A in t h e eye include p o t e n t i a l roles as b o t h o x i d a n t , a n t i - o x i d a n t an d free radical s c a v e n g e r p r e v e n t i n g * This work has been supported by Research Grant EY 04418 from The National Institute of Health (NEI), Bethesda, MD, U.S.A. t Correspondence address. 0014-4835/89/110873 + 13 $03.00/0
9 1989 Academic Press Limited
874
J. DIMATT10
oxidative damage and thereby maintaining an intraocular homeostasis Varma, Ets and Richards, 1977; Bhuyan and Bhuyan, 1977; Delamere, Paterson and Cotton, 1983; Giblin, McCready, K o d a m a and Reddy, 1984; Delamere and Williams, 1985; (Riley, Schwartz and Peters, 1986). We have recently presented in vivo evidence suggesting t h a t ascorbate enters aqueous humor of guinea-pigs via an active transport process (DiMattio and Streitman, 1986, 1987) and t h a t AA is the preferred form over dehydroascorbic acid with regard to transport into the eye. In addition, TLC studies indicated t h a t even with a pure dehydroascorbic step pulse delivered into blood, only labeled AA was found in aqueous humor suggesting t h a t AA is the only transported species of physiological importance to the guinea-pig eye. In contrast, parallel studies with rat indicated t h a t AA movement into aqueous humor was primarily via simple passive diffusion, as was that of dehydroascorbic acid. Moreover, TLC radiolabeled tracer studies revealed t h a t only the reduced AA was found in aqueous humor, suggesting t h a t dehydroascorbic acid was converted to AA before or during its entry into aqueous humor. This suggested that reduced AA is the physiologically important species also in the rat. No synthesis of AA is known to occur in lens (Kuck, 1970) yet its significant presence in lens of m a n y species (Varma, 1987) suggests t h a t aqueous humor and ciliary process are a likely route of passage. Serious questions remain, however, as to how the ascorbate moiety moves from blood to lens in the various species. Recent in vitro studies with whole lens of guinea-pig, bovine and humans suggested that mediated transport of dehydroascorbic acid occurred while uptake of reduced ascorbate was found to be negligible (Kern and Zolot. 1987). This left unclear how the lens achieves its high reduced AA and low dehydroascorbate levels. Moreover, other studies had reported t h a t both AA and dehydroascorbic acid were taken up at similar rates by lenses of rat and guinea-pig, indicating no carrier mediation for either species (Hughes and Hurley, 1970) and t h a t dehydroascorbic acid readily penetrated rabbit lenses in vitro (Shimuzu, 1965; van Heyningen, 1970). I t is the purpose of this work to help clarify the mechanism whereby ascorbic acid is brought into the lens. This was done by following the in vivo movement of labeled test molecules of AA, dehydroascorbic acid, L-glucose and 3-O-methyl-D-glucose from blood to aqueous humor, and subsequently into lens epithelium and lens interior or ' c o r t e x ' water compartments. Our work indicates that in the normal albino rat, which can produce AA, little or no carrier mediation at the level of the iris-ciliary process or the blood-aqueous barrier is present, but clear carrier mediation at the level of the aqueous-lens epithelium involving an active energy-requiring mechanism was observed. In contrast, little ascorbic acid was found to enter into the interior cortex water c o m p a r t m e n t in our experiments leading us to conclude that uptake by lens fiber cells is slow at best. Our work suggests t h a t the lens epithelium m a y be an important locus of ascorbic acid function, and t h a t special mechanisms exist in the rat lens epithelium which can meet this requirement.
2. M a t e r i a l s a n d M e t h o d s Current methods extend and improve those introduced previously (DiMattio, 1984). In short, the experimental procedure consisted of the introduction of a double label bolus of [3H]L-glucose (L-glu) and either [14C]L-ascorbic acid (AA), dehydroascorbic acid (DHA) or [14C]3-O-methyl-o-glucose (mD-glu) at time zero into the circulation of an anesthetized
ASCORBIC ACID TRANSPORT INTO LENS E P I T H E L I U M
875
(Nembutal : 50 mg kg -1, i.p.) and cannulated test rat (male Sprague-Dawley ; 250-300 g) and determining the decay in concentration with plasma samples taken periodically. At the prescribed end of the experimental period~ during which labeled molecules moved freely from blood to intraocular tissues, the animal was killed and samples of aqueous humor (10-20/el) were obtained. At this time the lens was removed and dissected to separate the capsuleepithelium from the interior cortex. The epithelium and cortex samples were quickly weighed and set to dry (60~ ; 24 hr). The following day the dry samples were weighed and returned to the oven until no further change in dry weight was observed (usually 3 days). The dried samples were then dissolved in tissue solubilizer (0"5 N quaternary ammonium hydroxide in toluene; Beckman BTS 450) and isotopic concentrations of labeled test species were determined (cpm/mg dry weight) via liquid scintillation spectrometry (Beckman LS4000) using Dimiscent scintillation cocktail (National Diagnostics). Water content for each of the samples was determined and the isotopic concentration expressed as cpm m1-1 of tissue water using appropriate values for tissue water content. The blood samples were centrifuged (600 g, 10 min) and 50/el samples of plasma were counted using Liquiscent scintillation cocktail (National Diagnostics). Double channel counting for 14C- and 3H-labels was used and the results were computer processed to correct for quenching and channel spillover and uniformly reported as dpm m1-1. The plasma data were fitted to a double exponential decay function with time. t (rain) from 0 to T min, when the animal was killed with an overdoes of sodium pentobarbital. Death was defined as the complete cessation of monitored cardiac activity and usually took less than 15 sec after lethal pentobarbital administration. This plasma decay function along with aqueous, lens epithelium and cortex concentration data was used to calculate transport rate constants which give a measure of how fast the concentrations of test molecules in a specific compartment change with time. Calculation of rate constants The plasma isotopic concentration data Cp(t) vs. time, t, for each test molecule were fitted graphically to a double exponential decay curve of the form : Cp = A + Be -bit + Ce-b2 t,
(1)
where A,B,C,b~ and be are determined constants. Constant A has been shown (DiMattio and Zadunaisky, 1981) to be the plasma concentration at t = oc, Cp(oc). Each experiment curve was normalized by dividing by a constant Cp(0), the plasma concentration at the onset of the experiment. The constants were determined graphically and via curve fitting using Asystant software (Macmillan Software Co., N.Y.). This program provides 'goodness of fit parameters' and resulted in plasma constants with a correlation coefficient of 0"992 or better. Thus, each experiment resulted in a specific plasma curve, with well-known defined constants, which were subsequently used to calculate appearance curves and entry rate constants for aqueous and lens compartments. Figure 1 illustrates the transport scheme used to derive appearance curves. For transport from blood plasma to aqueous humor, a simplified linear first-order system equation was used : dCA/dt = K~,Cp-KAo C,~. (2) T h i s equation indicates that the rate of change of concentration in aqueous humor is dependent simply on linear entry and exit rates with defined rate constants, KA~ and KAo. Note at t = oo. KA,/KAo = CA/Ca (oc). (3) For simple diffusion and carrier facilitated diffusion, the two adjacent compartments (e.g. plasma-aqueous) ideally come to a transport steady state with concentrations nearly equal and thus, CA/Cp(~) = 1"0 and Ka, = KAo = KA.
(4)
Another possibility is that C A / C e ( ~ ) > l'0 suggesting that some mechanism such as an active transport carrier is present which allows the accumulation of label to concentrations higher than those from which it originated.
876
J. D I M A T T I O
{ P)
Aqueous (A)
KA,
_
d~ ";';- : K,I C p - ~ , CA
IAqueous (AI
•/
Lens
- ~ : ~,C, -KEoC~
K,o
~-r : Kc, cr-KcoCc
FIo. 1. Transport schemes : from plasma (P) to aqueous (A) and from aqueous to lens epithelium (E) and lens interior 'cortex' compartments. Subscripts for rate constants, K (min -1) refer to destination compartment such as aqueous (A), epithelium (E) or cortex (C) with i refering to entry (in) and o, to exit (out) notations. Concentration, C, is dpm m1-1 and t, time (min-). W e limit o u r analysis to simple, carrier-facilitated diffusion a n d a c t i v e t r a n s p o r t m e c h a n i s m s for t h e t e s t molecules studied. U s i n g L-glucose as a control or passive m a r k e r allowed us to c o m p a r e results w i t h t h e o t h e r t e s t molecules a t least o n some r e l a t i v e basis in t h e same e x p e r i m e n t a l animal. As s h o w n previously, ( D i M a t t i o a n d Z a d u n a i s k y , 1981; D i M a t t i o , 1984), t h e a q u e o u s h u m o r c o n c e n t r a t i o n versus t i m e a p p e a r a n c e f u n c t i o n derived from e q n (1), a n d e q n (2) was s h o w n to be of t h e f o r m :
where
CA(t) = Sl(Anl + AA2 e-~n~ +An a e-b~' +An4 e-O~'),
(5)
S 1 - Cn ( ~ ) -- KA~
(6)
Cp(~) and
Kno
Anl = CA ( ~ ) / S~ = C e ( ~ ) = A
KnoB
KAoC
bl-KAo
b2-Kno
A n 2 = - - ~ - -
Knob
Ana - - -
Kno--b~
KnoC An4 = KAo_b2.
A
(7) (8)
(9)
(10)
A c o m p u t e r trial a n d error solution to eqns (4-6) allows for t h e e s t i m a t i o n of t r a n s p o r t c o n s t a n t s KA, a n d Kno using t h e d e t e r m i n e d p l a s m a c u r v e c o n s t a n t s of e q n (1), explicit a q u e o u s c o n c e n t r a t i o n d a t a a t a specific time, T, a n d some e s t i m a t e of t h e t r a n s p o r t s t e a d y state.
A S C O R B I C ACID T R A N S P O R T I N T O L E N S E P I T H E L I U M
877
Since lens epithelium is taken to be b a t h e d b y aqueous h u m o r and the concentration of labeled molecule in aqueous is represented by eqn (4), this equation is then used as the forcing function for transport into lens epithelium. Thus, the known constants of eqn (4), along with lens epithelium concentration d a t a at time, T, and some estimate of lens e p i t h e l i u m / a q u e o u s steady state are then used to determine the lens epithelium e n t r y and exit rate constants, Kz, and KEo, respectively. Transport rates into lens epithelium were taken as simple first order as was done for aqueous formation. The system equation is:
d C J d t = Ks~CA-Kso C~.
(11)
where E refers to ' Epithelium '. As a first approximation, the concentration of test molecule was assumed to be uniform t h r o u g h o u t the lens epithelium water c o m p a r t m e n t , with no distinction being made between intracellular or extracellular water. The equation t h a t describes the appearance and disappearance of labeled test molecule in lens epithelium with time is : C~(t) = 82(B~1 + BB~ e-KE~ + B.3 e-KA~ + B ~ e-blt + BB5 e-~2t),
(12)
S 2 - CA(~176 Kso
(13)
B m = C~ (oo)/S 2 = CA ( ~ )
(14)
where and
B,~-
AAd%~ AA3K~~ AAaK~~ KAo_K~~ +bl-~-~oo-+ b2-K~o AA,
(15)
AAd~.o -
(16)
B,3 = -
K.o-KA.
BB4 B~, -
AA~K.o
(17)
KEo-b~ AA4Kso
(18)
K~o-b2
Since lens cortex or interior tissue c o m p a r t m e n t is largely surrounded by epithelium we assume t h a t tracer molecules enter only from across the epithelium. Thus, in a parallel m a n n e r we used the concentration of test molecules as described in eqn (12) to be the forcing function for transport into a defined interior lens water c o m p a r t m e n t taken as 'cortex ', C. Again, transport into lens cortex is considered arising from simple first order rate expressions and the system equation is: dCc/dt = Kc, C~ -Keo Cc.
(19)
Using eqn (12) along with the known coefficients and constants, Laplace analysis was again used to derive an appearance and disappearance function ibr the ' c o r t e x ' or interior fiber cell c o m p a r t m e n t concentration, Cc(t ), given below: Cc(t ) = S3(Cc~+Cc~e-KC~176 where and
ga~
(20)
S 3 - Cc (oo) _ K~,
(21)
Cot = Cc ( ~ )/ S 3 = CE (oc)
(22)
C.(~)
K~o
Ce2 = B":'I(c~ + BsaKc~ .----_-7-B'4Ke~ BBsKc~ Keo-Kco KAo-Kco I-b~--Kco q b2-Kco Cca =
B.d;~o
eel
(23)
(24)
878
J. DIMATTIO cc4 - -
B.~co -
K~o-K~o
Cc~ -
B.4Kco -
-
K~o-bl
(25)
(26)
B.~K~o Cc~ -
Kco_b2.
(27)
Implied in our analysis are common simplifying assumptions concerning mixing and preservation of label on the molecule of interest. We note that small compartment volumes and convective circulation in aqueous humor, coupled with diffusion of small molecules, assists in mixing components of aqueous humor. I t is further noted that since nearly all the aqueous and complete lens tissue samples were taken, we are assured that at least uniform samples and average concentrations were used for rate constant calculations. Radio thin-layer chromatographic techniques were used to monitor the radiolabeled molecules in aqueous and tissue samples. Appropriate standards and labels were run on TLC plates and scanned with a Radiomatic Radioscanner RS with RTLC analysis software. Labeled L-glucose and 3-O-methyl-D-glucose were run on G-plates (Analtech, Newark, Delaware) using benzene : methanol : acetone : acetic acid (70 : 20 : 5 : 5) and ran with l~f's of 0-178 and 0"272, respectively, using methods outlined previously (DiMattio, 1988). Since ascorbic acid and dehydroascorbic acid are unstable in air a n d / o r water, all samples were spotted and analyzed under 100% nitrogen and confirmed using glass TLC silica gel H plates, custom prepared by Analtech with 6 % metaphosphoric acid to stabilize the ascorbic acid and prevent oxidation during analysis. The metaphosphoric acid H plates were run with acetonitrile :butyronitrile :water (66:33:2) and gave Rf's of 0-16 and 0"28 for ascorbic acid and dehydroascorbic, respectively.
3. R e s u l t s W a t e r c o n t e n t s of normal, male S p r a g u e - D a w l e y r a t s lens e p i t h e l i u m a n d lens ' c o r t e x ' c o m p a r t m e n t s were d e t e r m i n e d as functions of b o d y weight a n d age. W e n o t e t h a t for t r a n s p o r t e x p e r i m e n t s , r a t s used r a n g e d from 245 to 335 g, w i t h a m e a n of 296+_28 g. W e f o u n d t h a t lens e p i t h e l i u m % w a t e r c o n t e n t did n o t v a r y a p p r e c i a b l y w i t h r a t size a n d was t a k e n as 79"2 +_0"8 (n = 18) for calculations. Lens cortex w a t e r c o n t e n t was f o u n d to decrease w i t h age a n d weight. W e f o u n d 250 g r a t s h a d a lens cortex w a t e r c o n t e n t of 57-1 ___0"4 % (n = 9), 350 g rats, 55" 1 +- 0"3 % (n = 6), a n d 450 g rats, 54"2_ 0"3 % (n = 5). A p p r o p r i a t e values were used to calculate lens tissues c o n c e n t r a t i o n s in d p m m1-1 water. F i g u r e 2 illustrates compiled curves n o r m a l i z e d to the p l a s m a c o n c e n t r a t i o n a t t = 0, (Cpo), of c o n c e n t r a t i o n C vs. t (min). I t is n o t e w o r t h y t h a t A A leaves t h e p l a s m a f a s t e s t i n d i c a t i n g t h a t t h e b o d y r e m o v e s ascorbic acid efficiently from blood p r o b a b l y in an effort a t conservation. T a b l e I r e p o r t s m e a n curve fitting c o n s t a n t s of eqn (1) to i n d i v i d u a l curves o b t a i n e d in e x p e r i m e n t s . F i g u r e 3 r e p o r t s our c o m p i l e d results for a p p e a r a n c e of t h e v a r i o u s labeled molecules in aqueous h u m o r . N o t e t h a t [14C]3-O-methyl-D-glucose moves into aqueous h u m o r f a s t e s t of all t h e molecules t e s t e d a n d t h a t this n o n - m e t a b o l i z a b l e t e s t analog of D-glucose was p r e v i o u s l y shown to e n t e r b o t h aqueous a n d v i t r e o u s h u m o r in m a n n e r consistent w i t h f a c i l i t a t e d diffusion. These d a t a are consistent w i t h our p r e v i o u s r e p o r t s for whole lens (DiMattio, 1984). T a b l e I I r e p o r t s our results for p l a s m a to aqueous e n t r y r a t e c o n s t a n t calculations for the v a r i o u s molecules studied. N o t e t h a t we r e p o r t calculations based on 7 min a n d 13 min e x p e r i m e n t s . This was
ASCORBIC ACID T R A N S P O R T INTO LENS E P I T H E L I U M
879
1-000 o .... o ~ ~ =--= Q-- =
0.900 0.800
L-glucose L-Ascorbic acid 5 - O - m e t h y t - glucose dehydrocscorbic acid
0"700
g 0.600 "--~ 0.500
=
o.
LLL"
oo
0.2oo
J " ~ ; ~ ~ ~ - l ~
o.loo
~
.
~
~
0.000 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
IO
_
_
-1-
~
,. . . . . . . . . . . . . . . . . . . . 4.0 50
50
20
~
60
Time (rain)
Fie. 2. Plasma concentration vs. time (min) curves resulting from a bolus of labeled test molecular species introduced into a central plasma compartment at time 0. The concentrations are normalized to the concentration at time 0 or Cpo. Each curve represents a compilation from at least 12 experiments. TABLE [
Mean curve constants derived from a double exponential fit of plasma concentration vs. time (rain) data n*
A
lUll]L-glucose
(15)
0
[z4C]L-Ascorbic acid
(12)
0
[t4C]Dehydroascrobic acid
(7)
0
[14C]3-0-Methyl-D-glucose
(12)
0
B
C
0"383 0"617 +0"018 __+0'018 0"292 0"708 +0"077 • 0"366 0-634 -+0"073 -+0-073 0'456 0"544 -+0"008 -+0-008
b1
b~
0"0278 +0"0031 0'0475 -+0"0146 0"0409 -+0-0067 0"0195 __+0'0079
0"311 +0"039 0"488 +0"113 0"597 -+0'139 0"550 4-0'184
* n equals the number of experiments. Constants A, B, C, bl, b2 are defined by eqn (1) and are reported with S.D. below.
done to help s u p p o r t t h e n o t i o n t h a t t he m o d e l i n g of t r a n s p o r t was at least reasonable. We r e p o r t t h a t p l a s m a - a q u e o u s e n t r y r a tes for ascorbic acid were n o t found to be significantly different from those of L-glucose. This, along with ascorbic acid s a t u r a t i o n e x p e r i m e n t s with cold L-ascorbic acid an d t h e lack of a n y effect of glucose t r a n s p o r t inhibitors p h l o r e t i n (1 mM) a n d phloridzin (1 mM) on ascorbic acid m o v e m e n t , allowed us to suggest t h a t ascorbic acid e n t r y into aq u eo u s h u m o r of t h e r a t occurs p r i m a r i l y via simple passive diffusion (DiMattio, 1988). I n addition, we d e m o n s t r a t e d t h a t ascorbic acid enters a q u e o u s h u m o r unchanged, r e m a i n i n g in t h e reduced form, while d e h y d r o a s c o r b i c acid en t er ed or at least was f o u n d in a q u e o u s
880
J, DIMATTIO Rat
1'200 blOC 1,000 0'90C
I,
0,80C
o.7oc o.6ooi 0.500
i
s
0.400 0'500 0.200 0,100 0.000
.
0
.
.
I
i
.
IO
L-glucose ~ - - o Ascorbic acid , - - , 5-O-methyl-O-glucose A - - , Dehydro(:]scorbic acid " - - " .
.
I
20
.
.
.
.
I
50
.
.
.
.
l
40
.
.
.
.
l
.
.
.
50
.
I
60
.
.
.
.
I
.
.
70
.
.
l
.
.
i
,
80
I
90
Time (min)
Fro. 3. Illustrates the appearance of radiolabeled test molecules into aqueous. It is reported as the concentration in aqueous divided by the plasma concentration as a fucntion of tinie (min). TABLE I I
P l a s m a to aqueous humor entry rate constants, KA~
[all]L-glucose [14C]L-Ascorbicacid [14C]Dehydroascorbic acid [14C]3-O-MethylD-glucose
Ca/CP KA~ (1 per min)
CA/CP KAi (1 per min)
7-0 min
13"0 min
0"202+_0'021 0.0220+_@0018 0"352+_0"041 (9) (9) (13) 0-0286_+0.046 0"0236+,0"0029 0-561• 0'057 (5) (5) (8) 0-354_ 0"059 0"0357_+0.0049 0-693+ 0"07l (5) (5) (6) 0"671 +_0"071 0'102_+0"008 0"889_+0"059 (5) (5) (9)
0'0213+,0"0030 (13) 0"0243_+0"0038 (8) 0'0394 + 0"0058 (6) 0"0977+_0"006 (9)
Transport S.S. 1"0 1"0 !'0 1'0
o n l y in t h e r e d u c e d or ascorbic acid form (DiMattio, 1988). Thus, t h e fact t h a t d e h y d r o a s c o r b i c acid e n t r y r a t e s are a b o u t 40 % higher t h a n for ascorbie acid could reflect differences in passive p e r m e a b i l i t y p r o p e r t i e s between t h e two molecules or a m i n o r c o n t r i b u t i o n via carrier m e d i a t i o n . I n a n y event, 3-O-methyl-D-glucose e n t r y r a t e s into aqueous h u m o r were f o u n d to be b y far the f a s t e s t while L-glucose, ascorbic acid a n d d e h y d r o a s c o r b i c acid e n t e r e d into a q u e o u s h u m o r a t c o m p a r a b l y low r a t e s a n d p r i m a r i l y v i a passive diffusion. F i g u r e 4 r e p o r t s our c o m p i l e d e x p e r i m e n t a l results of t h e r a t i o of c o n c e n t r a t i o n in lens e p i t h e l i u m w a t e r c o m p a r t m e n t to t h a t f o u n d in aqueous a t v a r i o u s e x p e r i m e n t end times. Since e p i t h e l i u m to a q u e o u s h u m o r r a t i o s m u c h g r e a t e r t h a n 1"0 were seen in r e l a t i v e l y s h o r t t i m e periods, more t h a n simple or f a c i l i t a t e d diffusion is required t o e x p l a i n these results. I n c o n t r a s t , L-glucose a n d 3-O-methyl-D-glucose a p p r o a c h similar e q u i l i b r i u m c o n c e n t r a t i o n s r e l a t i v e to t h e circulating plasma. Also note t h a t
A S C O R B I C ACID T R A N S P O R T INTO L E N S E P I T H E L I U M
881
6,000 5.500
Tsl
5.000 4.500
.-:4 . 0 0 0 -~ E _= =la -~
5.500
/
5.000
xL
o ~ o -z~--
o L- glucose o ,Ascorbic acid z~ 3 - O - m e t h y t - D - g l u c o s e
2.500
f / / ~ ~j"~i~i0
~
0.000",~
~
0
,,, I0
~ _ ~ , 20
i,
~_,
50
.... 40
J .... 50
~ ..... 60
,,, 70
~ j ~ _ ~ 80
90
Time (rain)
FIG. 4. Illustrates the appearance of radiolabeled test molecules in the lens epithelium compartment. It is reported as epithelium concentration divided by the aqueous concentration. The number of experiments for each point is at least five while the n for the 7 and 13 min points are given in the tables.
the n-glucose analog reaches this equilibrium concentration significantly faster than the L-glucose, indicating a facilitating factor involved in transport. Figure 4 also illustrates t h a t our experimental results with ascorbic acid are significantly different and much faster. Concentration ratios as high as 5-0 were observed, indicating that an overall epithelium water c o m p a r t m e n t ascorbic acid concentration five times that of aqueous humor occurred within 20 min after introduction of the label bolus into blood. Thus, some mechanism is present which causes ascorbic acid to be accumulated in lens epithelium. This mechanism must be energy-requiring since aseorbic acid must be moved against a concentration gradient to achieve the concentrations noted by us. These factors suggest an active transport mechanism. Table I I I reports our rate constant calculations using 7- and 13-min experiments. T A ~ E III
Aqueous humor to lens epithelium entry rate constants, KE~
[all]L-glucose [I~C]L-Aseorbie acid [14C]3-O-MethylD-glucose
C J C a Ke, (1 per min)
C J C a Ks~ (1 per rain)
7'0 min
13"0 min
Transport S.S.
0-253+0-047 0.0283+__0"0031 0"411--+0"031 0'0261-+0'0028 (9) (9) (13) (13) 2-06 • 0-14 0-617-t-0"047 3"42_+0"21 0"672+0-054 (5) (5) (8) (8) 0.571 -+0-050 0-198__+0"015 0.803-+0-018 0.194+0-014 (5) (5) (9) (9)
1"0 5"0 1.0
* Values are listed as means _+s.D. with the number of experiments in parentheses below. 3O
E E R 49
882
J. DIMATTIO
Our calculated rate c o n s t a n t s indicate t h a t ascorbic acid e n t r y rates are more t h a n 20 times faster t h a n L-glucose, which was considered to be a comparable m a r k e r in size a n d molecular characteristics. These results indicate an active u p t a k e m e c h a n i s m for ascorbic acid b y lens epithelium. We note t h a t rate c o n s t a n t calculations utilized a n estimated t r a n s p o r t s t e a d y - s t a t e value reported in our results. A n y error in this e s t i m a t e would only m o d e r a t e l y effect our rate c o n s t a n t estimate, b u t would n o t alter results t h a t depend on actual d e t e r m i n e d c o n c e n t r a t i o n e n c o u n t e r e d a t specific times after the bolus i n t r o d u c t i o n .
IqO0
~
I.ooo
0.900
i
~
~
•
~ • T A
0-800
T/L
j o7oo
0.600
/I
e
L-glucose o - - o
0.2 0~ /
T/
AsCOrblcacid e - - e 5-0- rnethy[- D-glucose ~ - - a r
o
0
10
20
50
40 50 Time (rnin)
60
,
,
L
,
~
70
~
,
,
i
I
80
i
i
i
I
90
FIG. 5. Illustrates our data on the appearance ofradiolabeled molecules in the 'cortex' compartment. It is reported as cortex concentration divided by epithelium concentration. The number of experiments for each point is at least five. TABLE IV
Lens epithelium to cortex entry rate constants, Kc~
[all]L-glucose [x4C]L-Ascorbic acid [14C]3-O-Methyl])-glucose
Cc/C~ Ke, (1 per min)
Cc/CE Kci (1 per rain)
7"0 min
13"0 rain
Transport S.S.
0'092_+0"034 0"0101__0"0031 0"166_+0'053 0"0108__+0'036 (9) (9) (13) (13) 0'009_+0"0030"00178_+0"00041 0"020_+0"006 0"00167_+0-00048 (5) (5) (S) (S) 0"388_+0-049 0"175_+0-016 0"642_+0"084 0"180_+0"018 (5) (5) (9) (9)
1-0 1'0 1'0
* Values are listed as means-+s.D, with the number of experiments in parentheses below. Figure 5 reports our results with the lens interior or ' c o r t e x ' c o m p a r t m e n t . While ascorbic acid m o v e m e n t into lens e p i t h e l i u m was very fast, m o v e m e n t into the interior lens c o m p a r t m e n t was seen to be very slow. R a t e c o n s t a n t s calculated in p a r t
ASCORBIC ACID TRANSPORT INTO LENS EPITHELIUM
883
from the data of Fig. 5 are reported in Table IV and show in fact that entry into a mean interior lens water c o m p a r t m e n t is significantly slower even than that of Lglucose. Thus, if we assume L-glucose movement into lens cortex is via passive diffusion as the data suggest, then no carrier appears to be involved in moving ascorbic acid. In contrast, our data indicate t h a t the D-glucose analog enters lens quickly and efficiently at rates more than ten times those of L-glucose. Moreover, our results with mn-glu suggest that a facilitated transport mechanism is involved in glucose uptake by lens fiber cells since concentration ratios do not rise above 1.0. Thus, glucose transport into lens ' cortex' or interior fiber cells, is fast, but does not proceed against a concentration gradient ruling out active transport. 4. D i s c u s s i o n In a recent review on aseorbic acid and the eye, Varma has suggested that aqueous humor could act as a source for ascorbate for lens and cornea in species such as man, ox and rabbit which have high levels of ascorbate in aqueous humor (Varma, 1987). The rat, however, did not appear to fit in with this theory since little ascorbate was found in either plasma or aqueous humor. Recent work in this laboratory (DiMattio, 1986 ; 1988) with normal rats has revealed t h a t although low plasma ascorbate levels of 3"3+_0-8 mg per 100 ml (n -- 16) were detected, we have also found t h a t labeled aseorbic acid (MW 176) enters into aqueous humor at similar rates as the passive marker L-glucose (MW 180}, and t h a t the rate of entry into aqueous was not saturable with unlabeled ascorbate nor affected by the drugs phloretin (10 -3 M) and phloridzin (10 -3 M). These and other (Delamere and Williams, 1987) observations led us to conclude that ascorbic acid entry into aqueous humor was primarily via simple passive diffusion. Thus, aqueous humor ascorbate levels should be relatively low, reflecting low plasma levels. In fact, we found ascorbate levels to be even lower than in plasma, 2 " l + 0 . 5 m g per 100ml (n = 10), or about 60-70% of the circulating plasma level. This low ascorbate concentration in aqueous humor could well arise from uptake or utilization of ascorbate by intraocular tissue. In this report we found t h a t ascorbate is taken up by lens epithelium even against a concentration gradient. This observation could explain the low ascorbate levels found in aqueous by pointing to where at least some of the ascorbate had gone. A major finding in this work is t h a t while lens epithelium actively took up ascorbate, lens interior fiber cells in our ' c o r t e x ' compartment, apparently, did not. Lens fiber cells were seen to take up quickly and efficiently the non-metabolizable glucose analog [14C]3-O-methyl-D-glucose in a manner consistent with facilitated diffusion, but demonstrated little ability to take up ascorbate. Uptake rates for aseorbate were found to be significantly lower than even L-glucose. I t must be pointed out, however, t h a t we treat the whole lens interior as a single uniform c o m p a r t m e n t using average values, which m a y misrepresent the actual case and mask higher uptake levels by some regional fiber cells. We do not believe this to be the case, however, since L-glucose presumably moves into this interior c o m p a r t m e n t via passive diffusion and ascorbate should at least follow this pattern. We therefore suggest t h a t while ascorbate is actively taken up by lens epithelium, interior lens fiber cells demonstrate no interest in this molecule. This m a y be very important in attempting to understand where and what m a y be the essential functions of ascorbic acid which to date have remained somewhat elusive. In addition, the presence of capsule in our 'epithelium' c o m p a r t m e n t could also reflect a role for AA in capsule collagen formation. 30-2
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These findings appear to support the notion t h a t lens epithelium and fiber cell are not metabolically coupled. In addition, the facilitated D-glucose transporting system does not appear to facilitate ascorbic acid uptake as has been suggested to occur in other systems (Khatami, Li and Rockey, 1986). This is further complicated by other reports which suggest t h a t D-glucose and dehydroascorbic acid may share a common carrier (Bigley, Wirth, Layman, Riddle and Stankova, 1983). These issues remain unclear. Our in vivo system is complicated by geometry and complex intercellular relationships. I t m a y be that lens epithelium, by taking up ascorbate, does not allow it to proceed further to interior fiber cells to any great extent, while D-glucose more readily crosses this epithelium. Moreover, lens fiber cell D-glucose carriers, like neutrophils and fibroblasts, m a y require the dehydroascorbate moiety and do not facilitate AA movement into cells significantly. Hughes and Hurley (1970) have reported t h a t both ascorbic acid and dehydroascorbic acid were taken up by the lens of rat and guinea-pig at similar rates. We note, however, t h a t lenses had been previously isolated and stored in saline and, most importantly, no a t t e m p t was made to distinguish between epithelium and cortical uptake. In fact, no previous study of our knowledge has made this distinction. Without making any clear identification of differences in uptake between lens epithelium and interior cortical fiber cells, results could easily mask high or low uptake by these tissues. A more recent study with calf lens has suggested that only dehydroascorbic acid enters whole calf lens in a manner which appears to be related to facilitated glucose transport (Kern and Zolot, 1987). However, in this same study reported values of ascorbic acid and dehydroascorbic acid found in aqueous humor and lens indicate clearly that ascorbic acid has a significant presence in both tissue compartments while dehydroascorbic acid is nearly absent and amounts to only 3 - 1 0 % of the ascorbie acid concentrations. No direct comparison could be made, therefore, with this present work. I t has been previously noted that the presence of ascorbate in ocular tissue points to some as yet unknown function for this molecule. Our work further suggests t h a t even in a species which did not appear to have high ascorbate concentrations in ocular tissues, special mechanisms exist which bring this molecule into lens and more specifically into lens epithelium and likely for some purpose. ACKNOWLEDGMENTS The author is indebted to Mr Jack Streitman for expert technical assistance. The computer assistance of Mr Al Drayton is gratefully acknowledged. In addition, the author wishes to thank Dr V. Fisher for V.A. Hospital facilities without which this work would not have been possible. This work has been supported by Research Grant EY 04418 from the National Institute of Health (NEI), Bethesda, MD. REFERENCES Axelrod, J., Udenfriend, S. and Brodie, B. B. (1951). Ascorbic acid in aromatic hydroxylation. III. Effect of ascorbic acid on the hydroxylation of acetanilide, aniline and antipyrine in vivo. J. Pharmacol. Exp. Ther. l l l , 176-81. Bigley, R., Wirth, M., Layman, D., Riddle, M. and Stankova, L. (1983). Interaction between glucose and dehydroascorbate transport in human neutrofils and fibroblasts. Diabetes 32, 545-8. Bhuyan, K. C. and Bhuyan, D. K. {1977). Regulation of hydrogen peroxide in eye humors.
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Effect of 3-amino-lH-1,2,4-triazole on catalase and glutathione peroxidases of rabbit eye. Biochim. Biophys. Acta 497, 641 51. Bui-nguyen, M. H. (1985). Ascorbic acid and related compounds. Chromatographic Sci. 30, 267-301. Burns, J. J. (1975). Overview of ascorbic acid metabolism.. Ann. N.Y. Acad. Sci. 258, 5-7. Chattergee, I. B., Majnmder, A. K., Nandi, B. K. and Subramanian, N. (1975). Synthesis and some major functions of vitamin C in animals. Ann. N.Y. Acad. Sci. 258, 24-47. Delamere, N.A., Paterson, C.A. and Cotton, T . R . (1983). Lens cation transport and permeability changes following exposure to hydrogen peroxide. Exp. Eye Res. 37, 45-53. Delamere, N. A. and Williams, R . N . (1985). Detoxification of hydrogen peroxide by the rabbit iris-ciliary body. Exp. Eye Res. 40, 805-11. Delamere, N.A. and Williams, R . N . (1987). Comparative aspects of ascorbate in the ~queous humor. Invest. Ophthalmol. Vis. Sci. 28 (Suppl. 3), 74. DiMattio, J. A comparative study of ascorbic acid entry into aqueous and vitreous humors of the rat and guinea-pig. Invest Ophthalmol. Vis. Sci. (in press). DiMattio, J. and Streitman, J. (1986). In vivo ascorbic acid transport across ocular barriers and into retina of the normal rat. Invest: Ophthalmol. Vis. Sci. 27 (Suppl. 3), 350. DiMattio, J. and Streitman, J. (1987). A comparison of vitamin C and glucose transport across ocular barriers of rats and guipea-pigs. Invest. Ophthalmol. Vis. Sei. 28 (Suppl. 3), 74. Giblin, F. J., McCready, J. P., Kodama, T. and Reddy, V. N. (1984). A direct correlation between the levels of ascorbic acid and HeO 2 in aqueous humor. Exp. EyeRes. 38, 87 93. Hughes, R. E. and Hurley, R. J. (1970). In vitro uptake of ascorbic acid by the guinea-pig lens. Exp. Eye Res. 9, 175-80. Kern, H. L. and Zolot, S. L. (1987). Transport of vitamin C in the lens. Curt. Eye Res. 6, 885-96. Kh~tami, M., Li, W. and Roekey, J. H. (1986). Kinetics of ascorbate transport by cultured retinal capillary pericytes. Invest. Ophthalmol. Vis. Sci. 27, 1665-71. Kuck, J. F. g. (1970). Chemical constituents of the lens. In Biochemistry of the Eye. (Ed. Graymore, C. N.). Pp. 184 260. London: Academic Press. Levine, M. and Morita, K. (1985). Ascorbic acid in endocrine systems. Vit. Horm. 42, 2-64. Pirie, A. (1965). Glutathione peroxidase in lens and a source of hydrogen peroxide in aqueous humor. Biochem. J. 96, 244 53. Riley, M. V., Schwartz, C. A. and Peters, M. I. (1986). Interactions of ascorbate and H202 : implications for in vitro studies of lens and cornea. Curt. Eye Res. 5,207-16. Shimuzu, H. (1965). Experimental studies on the intake and the distribution of ascorbic acid in various tissues of the eye. Jpn. J. Ophthalmol. 9, 68-79. Staudinger, H. J., Krisch, K. and Leonhauser, S. (1961). Role of ascorbic acid in microsomal electron transport and the possible relationship to hydroxylation reactions. Ann. N.Y. Acad. Sci. 92, 195~207. Stetton, M. R. (1949). Some aspects of the metabolism of hydroxyproline studied with the aid of isotopic nitrogen. J. Biol. Chem. 181, 31 7. Van Heyningen, R. (1970). Ascorbic acid in the lens of the naphthalene-fed rabbit. Exp. Eye Res. 9, 38-48. Varma, S. D. (1987). Ascorbic acid and the eye with special reference to the lens. (Third Conference on Vitamin C.) Ann. N.Y. Acad. Sci. 498, 280-307. Varma, S. D., Bauer, S. A. and Richards, R. D. (1987). Hexose monophosphate shunt in rat lens: stimulation by vitamin C. Invest. Ophthalmol. Vis. Sci. 28, 1164-9. Varma, S. D., Ets, T. K. and Richards, R. D. (1977). Protection against superoxide radicals in rat lens. Ophthalmic Res. 9, 421-31. Zannoni, V.G. and La Du, B.N. (1960). Tyrosylurea resulting from inhibition of phydroxyphenylpyruvic acid oxidase in vitamin C-deficient guinea-pigs. J. Biol. Chem. 235(3), 2667-71.