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Some observations on the zinc metabolism of the rabbit lens
Eq.
Eye Res. (1984)
38, 497-507
Some Observations on the Zinc Metabolism the Rabbit Lens P. J. BENTLEY,
B. CHIN
AND
of
GRUBB
BARBARA
Departments of Anatomy, Physiological Sciences and Radiology, School of Veterinary Medicine, North Carolina State University, Raleigh, NC, 27606, U.S.A. and the Departments of Ophthalmology and Pharmacology, Mt Sinai School of Medicine, New York, NY, U.S.A. (Received 1 August
1983 and accepted 28 October 1983, New York)
Rabbits’ lenses contain about 100 pmol kg-i wet wt (145 pmol kg-i water) of zinc. This metal appears to be quite uniformly distributed throughout the organ and more than 90% is firmly incorporated into the tissue so as not to be readily exchangeable. The concentration of Zn in the aqueous and vitreous humors is about 10m5 M (one-fourth the concentration in the blood serum). Lenses incubated in vitro can accumulate Zn from solutions containing this concentration of the metal. This process is concentration-dependent and is increased following damage produced by metabolic inhibitors. The process probably involves diffusion and is increased in the presence of a low external calcium concentration and the Ca ionophore A 23187. Amino acids which are known to bind zinc did not influence its accumulation by the lens, with the exception of cystine which increased it. Accumulated Zn (using B6Zn as a tracer) was able to leave the lens, but this process was quite slow and was reduced by the presence of lanthanum and low Ca concentrations. It is suggested that Ca and Zn may share common binding sites in the tissue, and they could be utilizing the same channels to cross the cell membrane. Key words: lens: zinc; calcium; lanthanum; A 23187.
1. Introduction Zinc is an essential trace element in the body, where it has been shown to play a role in the actions of over 80 enzymes (see Underwood, 1977; Ulmer, 1977; Burnet, 1981; Prasad, 1979). Deficiency of this metal has been shown to influence the ability of cells to divide, especially those in epithelia. The crystalline lens is an epithelial tissue which contains substantial amounts of zinc (Galin, Nano and Hall, 1962; Swanson and Truesdale, 1971; Baldwin and Bentley, 1980). The lens also contains high concentrations of carbonic anhydrase which contain Zn, but the presence of this enzyme can only partly account for the quantity of this metal in the lens (Galin et al., 1962). The role of the remainder of the Zn is unknown, but it could be contributing to the structural integrity of the proteins which are involved in the maintenance of lens transparency. In an earlier investigation (Baldwin and Bentley, 1980) we used the amphibian lens (in vitro) as a tentative model for studying the Zn metabolism of the vertebrate lens. The present work utilizes the lens of the rabbit which, being a mammal, would be expected to mimic more closely the situation in the human. We have measured exchanges of Zn in this tissue and the effects of other minerals, amino acids and drugs on this process. The results now suggest that Zn and Ca may share common regulatory mechanisms and that physiological interactions between these minerals may occur. Correspondence should be addressed University. 4700 Hillsborough Street,
00194835/84/050497+11
$03.00/O
to: Dr P. J. Bentley, School Raleigh, NC 27606. T.S.A.
of Veterinary
@ 1984 Academic
Medicine,
Press Inc. (London)
N.C. State
Limited
2. Materials
and
Methods
Except where stated New Zealand white male rabbits weighing about 2.5 kg. about I2 weeks old, were used in the experiments. They WCTP rapidly killed by placing them in a conta.iner of carbon dioxide. The eyes were removed and the lenses (weighing about 300 mg) were extracted and placed in a solution containing 1314 mM NaU; 5.4 mM KU: 1.8 mM Ca gluconate ; 54 mM MgSO,; 1.2 mM NaH,PO,. 160 mrvr HEPES : 50 mM glucose and 66 g 1-i streptomycin sulfate at 35% We (McGahan. Clhin and Bentley, 1983) found that the lenses could maintain a reasonably stable ion (Na, K and Ca) content. in vitro in this solution. In the present experiments we found the mean Na content of six non-incubated rabbit lenses to be 11.3f66 meq kg-’ wet wt, while the K was 77k4.0 meq kg-’ wt. Following 24 hr incubation in the medium at 35°C the Na content of lenses was 14.61fr2.6 meq kg-’ wet wt and the K was 78 f 2.2 meq kg-i wet wt Zinc contamination of the incubation medium was almost undetectable by atomic absorption spect#rophotometrg (see below) and was about 4 x lo-’ mol 1-i. The lenses were each incubated in Erlenmeyer flasks containing 20 ml of the incubation medium ; the posterior side of the lens faced downwards. The flasks were suspended in a water bath at 35°C and gently rocked. The vitreous humor was not removed from the posterior side of the lens until the termination of the experiment. When appropriate the isotope, 6SZn (New England Nuclear, Boston, MA), was added to the medium, containing different concentrations of ‘cold’ Zn to give a concentration of isotope of about 0.2 @J ml-i. One lens of each pair usually served for an experimental procedure and the other as a control preparation. The zinc content of the lenses was determined by atomic absorption spectroscopy (Perkin Elmer, model 290B or 4000) and Na and K by flame photometry (Eppendorf). The lenses were ashed for 6 hr at 500°C’ in a muffle furnace. The residue was dissolved in 0.1 N HCI. Radioactivity was counted in a GAMMA counter (Beckman Biogamma, Model 2). Total accumulation of Zn was then calculated using the specific activity (counts ,amol-i) of the Zn in the external bathing solutions. The results are usually expressed as mmol kg-i wet wt following weighing of the lens to 1 mg on a Sartorius balance. The water content of some lenses was measured bv drying them at 110°C for 24 hr and weighing them to 62 mg on torsion balance (Federal Pa&). However, as only a minor portion of tissue zinc was found to be present ‘free’ in solution (see also Baldwin and Bentley, 1980) we usually expressed the results in terms of wet wt rather than water content. Swelling of the lenses was not observed except, in those which had been exposed to EDTA or metabolic inhibitors. With the latter this was as great as 20 y0 of the initial weight. The latter results were corrected for this change by reference to the weight of the paired control lens. Exchangeability of lenticular Zn was measured in the standard manner, which essentially utilizes a comparison of the isotopic (65Zn) specific activity of the total Zn in the external bathing media with that in the tissue following equilibration for a period in vitro. For such measurements the total concentration of the metal in the t.issue is required, and this level was determined by atomic absorption spectrophotometry in the contralateral lens incubated under identical conditions but with no isotope present. A reflection of the difference in the isotopic specific activity of the Zn in the tissue and bathing media is seen when the accumulation of external labelled Zn by the lens, at equilibrium. is expressed as a percentage of the total Zn present (Table III). Thus, if the calculated accumulation of the external Zn is only equivalent to loo/b of the total Zn present in the tissue at equilibrium then 9096 of the metal is considered to be inexchangeable with the isotopically labelled Zn under these conditions. The regional distribution of zinc in the lenses was measured after dissecting the frozen tissue with a scalpel. The outer part of the tissue was cut away in a series of tangential cuts so as to leave an inner medullary (‘nuclear’) portion which consisted of about 25’6 of the lens. The remaining intact ‘nuclear’ port.ion of the lens was analyzed. For the measurement of the uptake of Zn across the anterior side of the lens, the tissue was incubated in a ‘well-type’ chamber (McGahan. Chin and Bentley. 1983). ,phese were made from glass tubing and contained a central constriction narrow enough to hold the lens. The chamber was filled with the incubation medium and vertically suspended in a water bath maintained at 35°C. The lens, including some attached vitreous, was lowered into the top
ZR
METABOLISM
OF
499
LENS
of the chamber, anterior side facing upwards, and allowed to sink into the solution until it was retained by the constriction. Phenol red was placed in the upper solution so as to be able to observe that an appropriate seal had been made. The OSZn which was used as a tracer for Zn (about @2 &i ml-l) was always added to the top solution and at the end of the experiment a sample of fluid was taken from the lower solution; if the radioactivity exceeded 1 o/0 of that on the ‘hot’ side, the preparation was discarded. The volume of the solution on each side of the lens was about 10 ml and the height of the column of fluid holding the lens in place was about 10 cm. The top solution was gently stirred by bubbling it with air. The efflux of BSZn from the lens was measured after preloading the tissue for 3 hr in a solution containing 66Zn (about 0.2pCi ml-l). The accumulation of the isotope was quite reproducible at this time and approached what appeared to be an equilibrium level (Table IV). The lens was then transferred at specific time intervals through a series of isotope-free solutions. The results are expressed as the percentage remaining in the lens at the end of each time interval. The initial total, however, did not include that which was lost in the first 10 sec. which will include the excess in the film of fluid adhering to the lens. Iodoacetic acid, prostaglandin E, and p-chloromercuriphenylsulfonic acid (PCMPS) were obtained from the Sigma Chemical Company, St Louis, MO and A 23187 from Calbiochem. La Jolla. CA. 3. Results Zinc content of the rabbit lens and its bathing$uids The concentration of Zn in the rabbit’s lens is about 100 pmol kg-l wet wt (Table I) or 145 ,umol kg-l tissue water. There appeared to be no significant difference between the lenticular Zn levels in the male and female rabbits, nor between the two-week-old and U-week-old male animals. The lenses of the six-month-old male rabbits had a slightly higher concentration of Zn than the younger animals, but this level was within the range of variability observed in different batches of the 12-week-old male rabbits. TABLE
Concentrations
of zinc in lenses and ocular jluids Lens
(,umol kg-’ Male rabbits Two weeks old 12 weeks old Six months old Female rabbits 12 weeks old
I
wet wt)
96k4.3 (6) 105 + 1.6 (6) 116k3.2 (6)
of rabbits
Aqueous (pm01 1-l)
Vitreous (pm01 1-l)
Blood serum (pm01 1-l)
17++t 10.7 + 1.9 (6) 98*
14.1 k 6.9 (5)$ 96f@8 (11) 11.9*
37k7.5 (7) 44 + 8.0 (3)
97 k 3.0 (6)
Results are a8 means+s.~. for the number * Pooled samples from three rabbits. t Pooled samples from six rabbits. $ Ashed whole vitreous.
of determinations
given
in parentheses.
We measured the concentration of Zn in the ocular fluids and blood serum (Table I) of the 12-week-old males. The concentration of Zn in the aqueous and vitreous fluids was about 10m5 M, which is four times less than that in the serum. In order to see if there was any difference between the Zn concentrations in the inner ‘nuclear’ zone and the remainder of the organ we dissected freshly frozen lenses and analyzed the inner 25-30% of the organ. This nuclear region was found to have a concentration of 107 + 2.2 ,umol kg-’ wet wt (10) compared to 105 f 1.6 pmol kg-l wet wt in the paired intact lenses (means + S.E., number of experiments in parentheses).
P. J. BENTLEY
500
ESfects of external
ET
AL
Zn. OR the Na and K content of the lens in vitro
Rabbit lenses can maintain substantial gradients of Na and K concentration with the external media when incubated for 24 hr in vitro (Table II). When 1O-5 M Zn was added to the incubation media (similar to the concentration in the ocular fluids in vivo) the Na and K concentrations did not differ from those in the control lens. However, when the Zn concentration was increased to 1O-4 M there was a considerable gain (P < 0601 for the difference) of Na and loss of K, indicating that the functional integrity of the lens was not maintained and that this increased concentration of the metal was presumably toxic. TABLE
II
Effects of Zn on the Na and K content of rabbit lenses incubated 35°C (meq kg-’ wet wt) & Zn 10m6 M Control zll
lo+M
Contrql
23.6 k 1.8 244 &- 2.7 62.2 k 66 21.Ok2.2
Results are aa means f S.E. for six lenses. The controls zinc. For details of incubation see Methods.
Exchangeability
of lenticular
in vitro for 24 hr at
K 667 644 437 747 were paired
+ k + f
1.8 2.9 36 1.8 with
those exposed
to the added
zinc
Minerals may be present ‘free ’ in solution in tissue water or they may be sequestered in some manner which limits their mobility. When we incubated lenses for 24 hr in media containing no added Zn the lenticular concentration of this metal was 102 + 1% pmol kg-’ (6), which can be compared to a value of 107 + 2.2 ,amol kg-l wet wt in a group of non-incubated lenses from the same batch of rabbits. Little if any Zn appeared to be lost from lenses under these conditions. EDTA can remove Zn which is bound to protein imidazole and sulfhydryl groups, but not that present in metaloenzymes or bound to nucleic acids (see Underwood, 1977). However, exposure to a Ca-free media containing 2 mM EDTA for 24 hr did not significantly change the concentration of Zn in the lenses. The Zn level in the non-incubated lenses was 109 &- 3 pmol kg-’ wet wt (6) compared to 107 f 5 pmol kg-’ wet wt for the paired tissues incubated with the EDTA. In order to assess the exchangeability of the Zn in the tissue under more physiological circumstances we incubated lenses for 24 hr in solutions containing different concentrations of Zn and its isotope 65Zn (Table III). We compared the uptake of the Zn (using s5Zn as a tracer) with the total amount present in the paired lenses incubated with the same concentration of Zn but with no isotope present (see Methods). When the external concentration of Zn was 10P5 M, which is the same as that present in the ocular fluids in vivo, the accumulated external (labelled) Zn could only account for 84 o/o of the total Zn present in the lens. The remainder apparently could not exchange readily for external Zn under these particular conditions in vitro. We also incubated lenses in solutions containing lo+ M and 10P4 M Zn. In the latter solution a substantial net gain of Zn, as detected by atomic absorption spectrophotometry, occurred. However, in all these solutions about 100,umol kg-’ wet wt of the
Zh-
METABOLISM
OF
TABLE
The exchangeability
501
LENS
III
of external Zn (using E5Zn as a tracer) with rabbit lens. Incubation time 24 hr
‘cold’
Zn in the
Zinc concentration in external medium
I Zn accumulation from external solution (/cmol kg-* wet wt)
II Total ‘ cold ’ Zn in lenses paired to I (pmol kg-’ wet wt)
III ZninIaa percentage of II (= ‘exchange’)
io-’ io-& lo+
1.2kO.4 94 f 2.9 65k 1.4
117k2.2 112k3.9 162+ 11
1.03 84 40.1
M M* M
Results are as meansks.~. * The normal concentration
for six lenses. For further details see Methods. of zinc in the ocular fluids (see Table I).
lenticular Zn was not exchanged for the external Zn. The ‘free’ (exchangeable) Zn present in the tissue under in vitro conditions with the external Zn concentration similar to that in vivo, 10e5 M, would appear to be equivalent to less than 10 o/o of the total of this mineral present. Accumulation
of 2% by the lens in vitro
The accumulation of Zn from external bathing solutions was measured by using @+Zn as a tracer for the external Zn (Table IV). The metal was progressively taken up over a period of several hours. At a concentration of lop4 M Zn accumulation appeared to be maximal after about 3 hr, while at lower concentrations it continued to be taken up for a somewhat longer time. TABLE
The accumulation
of Zn (using
IV
s5Zn as a tracer) by the rabbit lens in vitro
(pm01kg-’ wet wt) Cont. of Zn in external media 10-s M 10-s M 10-4 31 Results
‘Dip’
5 set
0.06 + 0.01 1.06+0.12 12-2 &- 1.9
are as meansks.~.
1 hr
3 hr
5 hr
16 hr
24 hr
0.41+ 0.06 2.81 f 0.26 224 _+ 3.0
017+004 512+ 1.11 624k 8.4
045*0.10 CQ+ 1.10 64.9* 1.4
1.21 kO.27
1.20+@4 9.5 f 2.9 65.0 If: 1.4
for six lenses.
The ability of the lens to take up Zn across its anterior epithelial surface w&s measured by mounting the organs in divided glass chambers and using BbZn as a tracer (see Methods). When the bathing solution contained 10e5 M Zn the accumulation of Zn in 3 hr was 6-8 f 2.7 pmol kg-’ wet wt (6). In paired lenses incubated in the usual manner, so that the entire surface was exposed to the 06Zn, the uptake was 63 +O-38 pmol kg-’ wet wt. Zinc can thus be taken up across the anterior surface of the lens. The results were however quite variable and we do not know if this process
502
P. .l. BENTLEY
ET
.\I,
also occurs across the posterior surface. (The attached vitreous made it difficult to mount. the lenses in a satisfactory way for the latter type of measurement.) In order to see if the accumulated ‘j5Zn was distributed uniformly in the lens we measured the radioactivity present in an inner nuclear region, which in these experiments comprised 26 5 2.9 o/0 (6) of the total weight of the lens. The counts present in this segment of the tissue after 24 hr incubation in 10m5 M Zn only account for 2.1 -f0.8 o/0 of the total counts in the intact. lens. The metal thus apparently cannot, move freely within the lens. Effects of various
conditions
on the accumulation
of Zn by the lens
We measured the accumulation of Zn (using s5Zn as a tracer) by lenses bathed in media containing 10e5 M Zn (Table V). Exposure of the lens to metabolic inhibitors (iodoacetate plus cyanide), which produced pronounced swelling of the tissue, resulted in a large increase in the accumulation of Zn by the lens. The presence of other metal ions, those of mercury, cadmium, copper and lanthanum, did not alter the accumulation of the Zn. TABLEV
Effects of different
agents on 2% accumulation (using 66Zn as a tracer) by the rabbit lens in vitro for 5 hr (,umol/kg wet wt 5 hr)
Control Iodoacetate (5 mM) + CN (2 rnx) Hg2+ iO-5 M Cd2+ iO-5 M cl?+ 10-5 M La3+ 2 mzil Cal+ @1 miw 5rnM 10 miw A 23187 lO+ M Prostaglandin E, 10-S M Threonine 10m4 x Lysine 10-O M Histidine 10T4 M Glutamine lo-” M Cystine 10m4 M Cyst&e 10m4 M PCMPS 1O-4 x
Experimental
P for difference
42fl.2 95fl.l 5.2+ 1.2 5.9 + 0.9 6.3f0.6 62 + 0.2 6.5 & 0.6 54f@4 3.8fO.4
19.2k5.5 7.6f@6 55&0.8 7.7 + 1.1 65f06 11.6+ 1.3 3.5 * 0.3 20 * 0.4 19.2+ 1.1
< 0.05 n.s. n.s. n.s. n.s. < 0.01 < 0001 < 0001 < 0.001
59+e5 6.4 * @3 7.5 k 0.6 7.8+ 1.2 8.6 + 0.9 4,3+ 1.2 5.4kO.5 6.7 +O.Y
56f04 7.3f0‘9 6.9+ 1.6 9.5 * 1.5 8.5k1.3 8,8* 1.4 54kO.3 6.6 +_ 0.5
n.s. n.s. n.s. n.s. n.s. < 0.05 n.s. LS.
n.s. = not statistically significant (P > 0.05). Results are as means k S.E. of six pairs of lenses.
When the external Ca concentration was reduced to 10m4 accumulation of Zn was nearly doubled, while 10 mM Ca reduced it to about one-third of the control level (Table V). The calcium ionophore A 23187 produced a remarkable increase in Zn accumulation. In the rat intestine it has been suggested that prostaglandin E, can increase transport of Zn (Song and Adham, 1978). However, we could not show an effect of this autaeoid on Zn accumulation by the rabbit lens (Table V). Zinc can bind to several amino acids, including histidine, glutamine, threonine,
ZN
METABOLISM
OF
BO3
LER’S
lysine and cystine (Prasad and Oberleas, 1970). These amino acids have been identified in the aqueous humor of rabbits (Reddy, Rosenberg and Kinsey, 1961) but only cystine had any effect on the accumulation of Zn by the lenses; an increase was observed (Table V). The reduced form of cystine, cysteine, had no effect. As cystine is a disulfide compound which could interact with membrane sulfhydryl groups we tested the effects of PCMPS which can bind such moieties at the cell surface. However. it had no effect on the accumulation of Zn. Ejlux
of Zn from the lens
Lenses were incubated for 3 hr in a medium containing 10e5 M Zn and 65Zn. They were then removed from this solution and transferred through a series of incubation media in which no Zn was present (see Methods). The loss of the accumulated Zn was measured and expressed as percentage of total initial ‘ counts ’ or in ,umoles of Zn (based on the specific activity of the Zn in the bathing media containing the s5Zn). The isotope lost in the first 10 set rinse was not however used in the calculations as this mainly represented the excess incubation media on the lens surface, though also probably some of that which is present in the extracellular space. The rate of loss of the Zn Zn remained declined progressively and after 2.5 hr about 40 “/” of the accumulated in the tissue (Fig. 1). The decline in the rate of efflux of Zn was much greater than that of the calculated decrease in concentration of the accumulated Zn in the lens (see Discussion).
A
0
150 Time
(mid
FIG. 1. Efflux of Zn from the rabbit lens. Each point (O--O) 2 my lanthanum, (o-0) no lanthanum. For The procedure is described in the Methods and Results.
represents the meanCS.E. of six further data on these experiments
measurements see Table
VI.
In view of the previous experiments suggesting that Zn may move through similar pathways to calcium we measured the effects of lanthanum on the efflux of Zn. (La can block Ca-channels, both influx and efflux, in cell membranes and inhibits the activity of the Ca pump, see Weiss (1974).) The La resulted in a decline in the rate of efflux of Zn from the lens (Table VI) as also reflected by a reduction in the percentage loss of ‘counts’ by the tissue (Fig. 1). I !I EICR3x
I’. .I. I~EXTLEY TABLE
ET
dL.
\‘I
The rate of e$lux of Zn (using 65Zn as a, tracer) frowl rabbit lenses followi~ag for 3 hr in media contuinirbg IOPM %n* Zn efflux Time
period (min)
&l 5-10 20-30 6G-90
pnol
control
c:! m41 La
0.69 + 0.08 0~13~0014 004 & 0905 0010f00017
048fti0.5 0.09 + 0908 0.024 * @002 0.006 k OGOO7
kg-’
loading
wet wt min
P for difference < < < <
0.05 0.05 0.01 00.5
C’alculated cont. of 65Zn in lens PM 10 7 .M 44
As little of the Zn normally present in the lens was exchangeable the specific activity of the Zn was assumed to be the same as in the external ‘loading’ solution. * The external media for the efflux experiment contained no added Zn. For further details see Methods and Fig. 1, Results are as means f SE. for six experiments.
As changes in the external concentration of Ca (last section) have been shown to influence the accumulation of Zn by the lens we also measured the effects of this alkaline earth metal on the efflux of Zn. Lenses were loaded with Zn by incubating them for 3 hr in a solution of lop5 M Zn, containing 65Zn. The efflux was measured as described previously. The percentage counts which remained in the lens after 30 min were 38*6+ 15 (6) in control preparations (1.8 mM Ca) and 49.2 k2.4 in paired lenses exposed to a reduced, @1 mM, concentration of Ca (P < @Ol for the difference). Such a difference persisted throughout the 150 min of the efIlux measurement. When the concentration of the Ca was increased to 10 mM in the experimental medium no significant difference was observed ; for instance after 30 min the percentage remaining in the lens was 44.4k3.5 (6), while it was 37.4+ 2.3 in the control medium. 4. Discussion The rabbit lens contains about 100 ymol kg-’ wet wt of Zn, which is double that present in the amphibian lens (Baldwin and Bentley, 1980). This Zn appears to be distributed quite uniformly throughout the organ but, as also seen in the amphibian lens, less than 10 y. of the total is readily exchangeable. Most Zn in the lens is probably incorporated with the structural components of the tissue, especially proteins and nucleic acids. The aqueous and vitreous humors contain Zn at a concentration of 10e5 M, which is only one-fourth the concentration in the blood serum. Zinc is bound to plasma proteins (Prasad, 1979) and these may restrict its movement across the ciliary body into the aqueous humor. Zinc can slowly accumulate in the lens from solutions in which the concentration is similar to that in the rabbit’s vitreous and aqueous humors (10M5 M). The accumulation is far greater than that which can be accounted for by that present in the extracellular fluids of the lens, and it can be facilitated when integrity of the tissue is compromised by the presence of metabolic inhibitors. The restrictive capacity of the cell membrane is probably compromised in the latter circumstances, but there could also be an unmasking of chemical moieties to which the metal may be able to bind. Our estimate of the exchangeable fraction of the total Zn present in the lens suggests that the ‘free’ diffusible concentration in the tissue could be about 10m5 M
ZK
METABOLISM
;io5
Oh’ LENS
or less. The concentration in the rabbit ocular fluid is also 10e5 M suggesting, when the favorable gradient of electrical potential into the cells is taken into consideration, that Zn may move down an electrochemical gradient into the intracellular fluids. There is little information about the process of Zn entry into cells (see Underwood, 1977). The available information is derived principally from studies of its absorption across the intestine and its excretion in the urine (see Underwood, 1977; Foster, Aamodt, Henkin & Berman. 1979). This process could involve combination with ligands such as amino acids (Foster et al., 1979), and prostaglandin E, (Song and Adham, 1978). Zinc accumulation by the lens was not affected by such agents with the exception of cystine, which promoted accumulation. It is possible that the latter reflects binding of the Zn to the amino acid and its subsequent uptake by the lens. The uptake of Zn can also be inhibited in tissues by the presence of other metals such as copper and cadmium (see Underwood, 1977; Kingsley and Frazier, 1979). but such an effect was not seen in the present experiments on the rabbit lens. In the amphibian lens (Baldwin and Bentley, 1980) Cd decreased accumulation of Zn while Hg increased it. The pathway which Zn may follow into the lenticular cells is unknown, but it was of special interest to observe that the accumulation by the lens was enhanced when the external Ca concentration was reduced. Uptake was also increased in the presence of the Ca ionophore A 23 187. The efflux of Ca from cells can be inhibited by lanthanum, and this effect has also been observed in the rabbit lens (Hightower, Leverenz and Reddy, 1980; McGahan et al., 1983). The efflux of Zn from the rabbit lens was also reduced by La. However, the accumulation of Zn by the rabbit lens was not changed in the presence of external La suggesting that like Ca in other tissues (see Weiss, 1974) both influx and efflux of the metal may be inhibited. In the amphibian l.ens, only the influx of Zn appeared to be blocked (Baldwin and Bentley, 1980). It is thus attractive to consider the possibility that Zn and Ca follow similar pathways in and out of the lenticular cells. These sites could be diffusion channels and even involve the activity of an active pump mechanism such as a ‘Ca pump’. In the instance of Ca both types of process are known to be inhibited by La (see Weiss, 1974). Zinc is known to antagonize the effects of calcium and they may compete for similar binding sites (Ciofalo and Thomas, 1965; Prasad, 1979). The observed inhibitory effect of low external Ca concentrations on the efflux of Zn from the lens would be consistent with a reduced exchange of external Ca for bound Zn. The rate of efflux of accumulated Zn from the lens declined progressively and could not be readily related to the concentration of the metal in the tissue, if it is assumed to be present ‘free’ in solution. This inconsistency may reflect binding of the metal to tissue components, the presence of an ‘active pump’ mechanism, and/or interference due t’o the layered structural arrangement of the lens cells. Interference to solute movements due to the latter has been noted by Schultz and Curran (1970). The preceding observations on the Zn content of the rabbits’ lens and ocular fluids, and the behavior of the metal under in vitro conditions can be used to suggest a possible pattern of Zn metabolism in the lens in viva. The concentration of Zn in the aqueous and vitreous humors is adequate to assure access of the metal into the peripheral lens cells. The Zn can enter the lens across its anterior epithelial surface (possible uptake from the posterior side has not been excluded) and passes across the plasma membrane into the cells. This process is normally somewhat restricted and may involve specific pathways which may be synonymous with Ca channels and Ca-binding sites. The amino acids present in the ocular fluids probably do not interfere with this 1%”
506
P. .I. 13EKTI,E\’
ET
AI,.
process. with the possible exception of’ cystine, which could facilitatr uptake of the met,al. Once inside the cells Zn may be bound, incorporated into tissue components or remain relatively free to move out of the lens, possibly along the same pathways by which it entered the tissue. or through an active ‘pump’ mechanism. The rate of Zn accumulation by the lens may principally reflect it,s concentration in t.he ocular fluids: which is much less than that in the plasma. The ultimate control of the Zn metabolism of the lens may be due to the processes that, secrete the ocular fluids, especially the activity of the ciliary body. There
is little
specific
information
about
the
possible
role
of Zn in maintaining
lens
t’ransparency. It has been observed that senile human cataractous lenses have low levels of Zn (Swanson and Truesdale. 1971). A Zn-deficient diet has also been shown to result in cataracts in growing Rainbow trout (Ketola, 1979). Zinc is an essential trace element, but whether or not it has a special role in the maintenance of lens transparency is unknown. However. it could be involved in the stabilization of the structure of lens proteins and the removal of toxic free radicals from the tissue (Editorial, 1978). ACKNOWLEDGMENTS This work was supported by National We are grateful to Mr John Morris for
Institutes technical
of Health assistance.
grants
EY 01278
and EY 04755.
REFERENCES Baldwin, G. A. and Bentley, P. J. (1980). The zinc metabolism of the amphibian lens. Exp. Eye Res. 30, 333-43. Burnet. F. M. (1981). A possible role of zinc in the pathology of dementia. Lancet i, 18&8. Chvapil, M., Elias. S. L.. Ryan, J. N. and Zukoski, C. F. (1972). Pathophysiology of zinc. International Review of Neurobiology, Suppl. 1 (Ed. Pfeiffer, C. C.). Pp. 15-24. Academic Press, New York and London. Ciofalo, F. R. and Thomas, L. J. (1965). The effects of zinc on contractility, membrane potentials, and cation content of rat atria. J. Gen. Physiol. 48, 825-39. Editorial (1978). A radical approach to zinc. Lancet i, 191-2. Foster, D. M.. Aamodt, R. L., Henkin. R. I. and Berman, M. (1979). Zinc metabolism in humans: a kinetic model. Am. J. Physiol. 237, R34&9. Galin. M. A., Nano, H. D. and Hall. T. (1962). Ocular zinc concentration. Invest. OphthaZmoZ. 1. 142-8. Hightower, K. It., Leverenz, V. and Reddy. V. N. (1980). Calcium transport in the lens. Invest. Ophthalmol. Vis. Sci. 19, 105%66. Ketola, H. G. (1979). Influence of dietary zinc on cataracts in rainbow trout. J. Nutr. 109, 96.59. Kingsley, B. S. and Frazier, J. M. (1979). Cadmium transport in isolated perfused rat liver: zinc-cadmium competition. Am. d. Physiol. 236. C 13S43. McGahan, M. C., Chin, B. and Bentley. P. .J. (1983). Calcium metabolism of the rabbit lens. Exp. Eye Res. 37, 57-66. Prasad, A. S. (1979). Clinical, biochemical, and pharmacological role of zinc. Attn. Rut’. Pharmacol. Toxicol. 20, 393426. Prasad. A. S. and Oberleas, D. (1970). Binding of zinr to amino acids and serum proteins in vitro. J. Lab. C&n. Med. 76. 416-25. V. E. (1961). Steady state distribution of free Reddy, D. V. N., Rosenberg, C. and Kinsey. amino acids in the aqueous humours. vitreous body and plasma of the rabbit. Exp. Eye Res. 1, 175-81. Schultz, S. G. and Curran, P. F. (1970). Coupled transport of sodium and organic solutes. Physiol. Rev. 50, 637-718. Song, M. K. and Adham, N. F. (1978). Role of prostaglandin E, in zinc absorption in the rat. Am. J. Physiol. 234, E94-105.
ZS
METABOLISM
OF
LESS
507
Swanson, A. A. and Truesdale, A. W. (1971). Elemental analysis in normal and cataractous human lens tissue. Biochem. Biophys. Res. Commun. 45, 1488-96. Ulmer. D. D. (1977). Trace elements. New Engl. J. Med. 297, 318-21. Underwood, E. J. (1977). Trace elements in human and animal nutrition. fourth edition. Pp. 19&242. Academic Press, New York. San Francisco, London. Weiss, G. B. (1974). Cellular pharmacology of lanthanum. ,4nn. Rev. Pharmacol. 14. 343-5-C.
Eye Res. (1984)
38, 497-507
Some Observations on the Zinc Metabolism the Rabbit Lens P. J. BENTLEY,
B. CHIN
AND
of
GRUBB
BARBARA
Departments of Anatomy, Physiological Sciences and Radiology, School of Veterinary Medicine, North Carolina State University, Raleigh, NC, 27606, U.S.A. and the Departments of Ophthalmology and Pharmacology, Mt Sinai School of Medicine, New York, NY, U.S.A. (Received 1 August
1983 and accepted 28 October 1983, New York)
Rabbits’ lenses contain about 100 pmol kg-i wet wt (145 pmol kg-i water) of zinc. This metal appears to be quite uniformly distributed throughout the organ and more than 90% is firmly incorporated into the tissue so as not to be readily exchangeable. The concentration of Zn in the aqueous and vitreous humors is about 10m5 M (one-fourth the concentration in the blood serum). Lenses incubated in vitro can accumulate Zn from solutions containing this concentration of the metal. This process is concentration-dependent and is increased following damage produced by metabolic inhibitors. The process probably involves diffusion and is increased in the presence of a low external calcium concentration and the Ca ionophore A 23187. Amino acids which are known to bind zinc did not influence its accumulation by the lens, with the exception of cystine which increased it. Accumulated Zn (using B6Zn as a tracer) was able to leave the lens, but this process was quite slow and was reduced by the presence of lanthanum and low Ca concentrations. It is suggested that Ca and Zn may share common binding sites in the tissue, and they could be utilizing the same channels to cross the cell membrane. Key words: lens: zinc; calcium; lanthanum; A 23187.
1. Introduction Zinc is an essential trace element in the body, where it has been shown to play a role in the actions of over 80 enzymes (see Underwood, 1977; Ulmer, 1977; Burnet, 1981; Prasad, 1979). Deficiency of this metal has been shown to influence the ability of cells to divide, especially those in epithelia. The crystalline lens is an epithelial tissue which contains substantial amounts of zinc (Galin, Nano and Hall, 1962; Swanson and Truesdale, 1971; Baldwin and Bentley, 1980). The lens also contains high concentrations of carbonic anhydrase which contain Zn, but the presence of this enzyme can only partly account for the quantity of this metal in the lens (Galin et al., 1962). The role of the remainder of the Zn is unknown, but it could be contributing to the structural integrity of the proteins which are involved in the maintenance of lens transparency. In an earlier investigation (Baldwin and Bentley, 1980) we used the amphibian lens (in vitro) as a tentative model for studying the Zn metabolism of the vertebrate lens. The present work utilizes the lens of the rabbit which, being a mammal, would be expected to mimic more closely the situation in the human. We have measured exchanges of Zn in this tissue and the effects of other minerals, amino acids and drugs on this process. The results now suggest that Zn and Ca may share common regulatory mechanisms and that physiological interactions between these minerals may occur. Correspondence should be addressed University. 4700 Hillsborough Street,
00194835/84/050497+11
$03.00/O
to: Dr P. J. Bentley, School Raleigh, NC 27606. T.S.A.
of Veterinary
@ 1984 Academic
Medicine,
Press Inc. (London)
N.C. State
Limited
2. Materials
and
Methods
Except where stated New Zealand white male rabbits weighing about 2.5 kg. about I2 weeks old, were used in the experiments. They WCTP rapidly killed by placing them in a conta.iner of carbon dioxide. The eyes were removed and the lenses (weighing about 300 mg) were extracted and placed in a solution containing 1314 mM NaU; 5.4 mM KU: 1.8 mM Ca gluconate ; 54 mM MgSO,; 1.2 mM NaH,PO,. 160 mrvr HEPES : 50 mM glucose and 66 g 1-i streptomycin sulfate at 35% We (McGahan. Clhin and Bentley, 1983) found that the lenses could maintain a reasonably stable ion (Na, K and Ca) content. in vitro in this solution. In the present experiments we found the mean Na content of six non-incubated rabbit lenses to be 11.3f66 meq kg-’ wet wt, while the K was 77k4.0 meq kg-’ wt. Following 24 hr incubation in the medium at 35°C the Na content of lenses was 14.61fr2.6 meq kg-’ wet wt and the K was 78 f 2.2 meq kg-i wet wt Zinc contamination of the incubation medium was almost undetectable by atomic absorption spect#rophotometrg (see below) and was about 4 x lo-’ mol 1-i. The lenses were each incubated in Erlenmeyer flasks containing 20 ml of the incubation medium ; the posterior side of the lens faced downwards. The flasks were suspended in a water bath at 35°C and gently rocked. The vitreous humor was not removed from the posterior side of the lens until the termination of the experiment. When appropriate the isotope, 6SZn (New England Nuclear, Boston, MA), was added to the medium, containing different concentrations of ‘cold’ Zn to give a concentration of isotope of about 0.2 @J ml-i. One lens of each pair usually served for an experimental procedure and the other as a control preparation. The zinc content of the lenses was determined by atomic absorption spectroscopy (Perkin Elmer, model 290B or 4000) and Na and K by flame photometry (Eppendorf). The lenses were ashed for 6 hr at 500°C’ in a muffle furnace. The residue was dissolved in 0.1 N HCI. Radioactivity was counted in a GAMMA counter (Beckman Biogamma, Model 2). Total accumulation of Zn was then calculated using the specific activity (counts ,amol-i) of the Zn in the external bathing solutions. The results are usually expressed as mmol kg-i wet wt following weighing of the lens to 1 mg on a Sartorius balance. The water content of some lenses was measured bv drying them at 110°C for 24 hr and weighing them to 62 mg on torsion balance (Federal Pa&). However, as only a minor portion of tissue zinc was found to be present ‘free’ in solution (see also Baldwin and Bentley, 1980) we usually expressed the results in terms of wet wt rather than water content. Swelling of the lenses was not observed except, in those which had been exposed to EDTA or metabolic inhibitors. With the latter this was as great as 20 y0 of the initial weight. The latter results were corrected for this change by reference to the weight of the paired control lens. Exchangeability of lenticular Zn was measured in the standard manner, which essentially utilizes a comparison of the isotopic (65Zn) specific activity of the total Zn in the external bathing media with that in the tissue following equilibration for a period in vitro. For such measurements the total concentration of the metal in the t.issue is required, and this level was determined by atomic absorption spectrophotometry in the contralateral lens incubated under identical conditions but with no isotope present. A reflection of the difference in the isotopic specific activity of the Zn in the tissue and bathing media is seen when the accumulation of external labelled Zn by the lens, at equilibrium. is expressed as a percentage of the total Zn present (Table III). Thus, if the calculated accumulation of the external Zn is only equivalent to loo/b of the total Zn present in the tissue at equilibrium then 9096 of the metal is considered to be inexchangeable with the isotopically labelled Zn under these conditions. The regional distribution of zinc in the lenses was measured after dissecting the frozen tissue with a scalpel. The outer part of the tissue was cut away in a series of tangential cuts so as to leave an inner medullary (‘nuclear’) portion which consisted of about 25’6 of the lens. The remaining intact ‘nuclear’ port.ion of the lens was analyzed. For the measurement of the uptake of Zn across the anterior side of the lens, the tissue was incubated in a ‘well-type’ chamber (McGahan. Chin and Bentley. 1983). ,phese were made from glass tubing and contained a central constriction narrow enough to hold the lens. The chamber was filled with the incubation medium and vertically suspended in a water bath maintained at 35°C. The lens, including some attached vitreous, was lowered into the top
ZR
METABOLISM
OF
499
LENS
of the chamber, anterior side facing upwards, and allowed to sink into the solution until it was retained by the constriction. Phenol red was placed in the upper solution so as to be able to observe that an appropriate seal had been made. The OSZn which was used as a tracer for Zn (about @2 &i ml-l) was always added to the top solution and at the end of the experiment a sample of fluid was taken from the lower solution; if the radioactivity exceeded 1 o/0 of that on the ‘hot’ side, the preparation was discarded. The volume of the solution on each side of the lens was about 10 ml and the height of the column of fluid holding the lens in place was about 10 cm. The top solution was gently stirred by bubbling it with air. The efflux of BSZn from the lens was measured after preloading the tissue for 3 hr in a solution containing 66Zn (about 0.2pCi ml-l). The accumulation of the isotope was quite reproducible at this time and approached what appeared to be an equilibrium level (Table IV). The lens was then transferred at specific time intervals through a series of isotope-free solutions. The results are expressed as the percentage remaining in the lens at the end of each time interval. The initial total, however, did not include that which was lost in the first 10 sec. which will include the excess in the film of fluid adhering to the lens. Iodoacetic acid, prostaglandin E, and p-chloromercuriphenylsulfonic acid (PCMPS) were obtained from the Sigma Chemical Company, St Louis, MO and A 23187 from Calbiochem. La Jolla. CA. 3. Results Zinc content of the rabbit lens and its bathing$uids The concentration of Zn in the rabbit’s lens is about 100 pmol kg-l wet wt (Table I) or 145 ,umol kg-l tissue water. There appeared to be no significant difference between the lenticular Zn levels in the male and female rabbits, nor between the two-week-old and U-week-old male animals. The lenses of the six-month-old male rabbits had a slightly higher concentration of Zn than the younger animals, but this level was within the range of variability observed in different batches of the 12-week-old male rabbits. TABLE
Concentrations
of zinc in lenses and ocular jluids Lens
(,umol kg-’ Male rabbits Two weeks old 12 weeks old Six months old Female rabbits 12 weeks old
I
wet wt)
96k4.3 (6) 105 + 1.6 (6) 116k3.2 (6)
of rabbits
Aqueous (pm01 1-l)
Vitreous (pm01 1-l)
Blood serum (pm01 1-l)
17++t 10.7 + 1.9 (6) 98*
14.1 k 6.9 (5)$ 96f@8 (11) 11.9*
37k7.5 (7) 44 + 8.0 (3)
97 k 3.0 (6)
Results are a8 means+s.~. for the number * Pooled samples from three rabbits. t Pooled samples from six rabbits. $ Ashed whole vitreous.
of determinations
given
in parentheses.
We measured the concentration of Zn in the ocular fluids and blood serum (Table I) of the 12-week-old males. The concentration of Zn in the aqueous and vitreous fluids was about 10m5 M, which is four times less than that in the serum. In order to see if there was any difference between the Zn concentrations in the inner ‘nuclear’ zone and the remainder of the organ we dissected freshly frozen lenses and analyzed the inner 25-30% of the organ. This nuclear region was found to have a concentration of 107 + 2.2 ,umol kg-’ wet wt (10) compared to 105 f 1.6 pmol kg-l wet wt in the paired intact lenses (means + S.E., number of experiments in parentheses).
P. J. BENTLEY
500
ESfects of external
ET
AL
Zn. OR the Na and K content of the lens in vitro
Rabbit lenses can maintain substantial gradients of Na and K concentration with the external media when incubated for 24 hr in vitro (Table II). When 1O-5 M Zn was added to the incubation media (similar to the concentration in the ocular fluids in vivo) the Na and K concentrations did not differ from those in the control lens. However, when the Zn concentration was increased to 1O-4 M there was a considerable gain (P < 0601 for the difference) of Na and loss of K, indicating that the functional integrity of the lens was not maintained and that this increased concentration of the metal was presumably toxic. TABLE
II
Effects of Zn on the Na and K content of rabbit lenses incubated 35°C (meq kg-’ wet wt) & Zn 10m6 M Control zll
lo+M
Contrql
23.6 k 1.8 244 &- 2.7 62.2 k 66 21.Ok2.2
Results are aa means f S.E. for six lenses. The controls zinc. For details of incubation see Methods.
Exchangeability
of lenticular
in vitro for 24 hr at
K 667 644 437 747 were paired
+ k + f
1.8 2.9 36 1.8 with
those exposed
to the added
zinc
Minerals may be present ‘free ’ in solution in tissue water or they may be sequestered in some manner which limits their mobility. When we incubated lenses for 24 hr in media containing no added Zn the lenticular concentration of this metal was 102 + 1% pmol kg-’ (6), which can be compared to a value of 107 + 2.2 ,amol kg-l wet wt in a group of non-incubated lenses from the same batch of rabbits. Little if any Zn appeared to be lost from lenses under these conditions. EDTA can remove Zn which is bound to protein imidazole and sulfhydryl groups, but not that present in metaloenzymes or bound to nucleic acids (see Underwood, 1977). However, exposure to a Ca-free media containing 2 mM EDTA for 24 hr did not significantly change the concentration of Zn in the lenses. The Zn level in the non-incubated lenses was 109 &- 3 pmol kg-’ wet wt (6) compared to 107 f 5 pmol kg-’ wet wt for the paired tissues incubated with the EDTA. In order to assess the exchangeability of the Zn in the tissue under more physiological circumstances we incubated lenses for 24 hr in solutions containing different concentrations of Zn and its isotope 65Zn (Table III). We compared the uptake of the Zn (using s5Zn as a tracer) with the total amount present in the paired lenses incubated with the same concentration of Zn but with no isotope present (see Methods). When the external concentration of Zn was 10P5 M, which is the same as that present in the ocular fluids in vivo, the accumulated external (labelled) Zn could only account for 84 o/o of the total Zn present in the lens. The remainder apparently could not exchange readily for external Zn under these particular conditions in vitro. We also incubated lenses in solutions containing lo+ M and 10P4 M Zn. In the latter solution a substantial net gain of Zn, as detected by atomic absorption spectrophotometry, occurred. However, in all these solutions about 100,umol kg-’ wet wt of the
Zh-
METABOLISM
OF
TABLE
The exchangeability
501
LENS
III
of external Zn (using E5Zn as a tracer) with rabbit lens. Incubation time 24 hr
‘cold’
Zn in the
Zinc concentration in external medium
I Zn accumulation from external solution (/cmol kg-* wet wt)
II Total ‘ cold ’ Zn in lenses paired to I (pmol kg-’ wet wt)
III ZninIaa percentage of II (= ‘exchange’)
io-’ io-& lo+
1.2kO.4 94 f 2.9 65k 1.4
117k2.2 112k3.9 162+ 11
1.03 84 40.1
M M* M
Results are as meansks.~. * The normal concentration
for six lenses. For further details see Methods. of zinc in the ocular fluids (see Table I).
lenticular Zn was not exchanged for the external Zn. The ‘free’ (exchangeable) Zn present in the tissue under in vitro conditions with the external Zn concentration similar to that in vivo, 10e5 M, would appear to be equivalent to less than 10 o/o of the total of this mineral present. Accumulation
of 2% by the lens in vitro
The accumulation of Zn from external bathing solutions was measured by using @+Zn as a tracer for the external Zn (Table IV). The metal was progressively taken up over a period of several hours. At a concentration of lop4 M Zn accumulation appeared to be maximal after about 3 hr, while at lower concentrations it continued to be taken up for a somewhat longer time. TABLE
The accumulation
of Zn (using
IV
s5Zn as a tracer) by the rabbit lens in vitro
(pm01kg-’ wet wt) Cont. of Zn in external media 10-s M 10-s M 10-4 31 Results
‘Dip’
5 set
0.06 + 0.01 1.06+0.12 12-2 &- 1.9
are as meansks.~.
1 hr
3 hr
5 hr
16 hr
24 hr
0.41+ 0.06 2.81 f 0.26 224 _+ 3.0
017+004 512+ 1.11 624k 8.4
045*0.10 CQ+ 1.10 64.9* 1.4
1.21 kO.27
1.20+@4 9.5 f 2.9 65.0 If: 1.4
for six lenses.
The ability of the lens to take up Zn across its anterior epithelial surface w&s measured by mounting the organs in divided glass chambers and using BbZn as a tracer (see Methods). When the bathing solution contained 10e5 M Zn the accumulation of Zn in 3 hr was 6-8 f 2.7 pmol kg-’ wet wt (6). In paired lenses incubated in the usual manner, so that the entire surface was exposed to the 06Zn, the uptake was 63 +O-38 pmol kg-’ wet wt. Zinc can thus be taken up across the anterior surface of the lens. The results were however quite variable and we do not know if this process
502
P. .l. BENTLEY
ET
.\I,
also occurs across the posterior surface. (The attached vitreous made it difficult to mount. the lenses in a satisfactory way for the latter type of measurement.) In order to see if the accumulated ‘j5Zn was distributed uniformly in the lens we measured the radioactivity present in an inner nuclear region, which in these experiments comprised 26 5 2.9 o/0 (6) of the total weight of the lens. The counts present in this segment of the tissue after 24 hr incubation in 10m5 M Zn only account for 2.1 -f0.8 o/0 of the total counts in the intact. lens. The metal thus apparently cannot, move freely within the lens. Effects of various
conditions
on the accumulation
of Zn by the lens
We measured the accumulation of Zn (using s5Zn as a tracer) by lenses bathed in media containing 10e5 M Zn (Table V). Exposure of the lens to metabolic inhibitors (iodoacetate plus cyanide), which produced pronounced swelling of the tissue, resulted in a large increase in the accumulation of Zn by the lens. The presence of other metal ions, those of mercury, cadmium, copper and lanthanum, did not alter the accumulation of the Zn. TABLEV
Effects of different
agents on 2% accumulation (using 66Zn as a tracer) by the rabbit lens in vitro for 5 hr (,umol/kg wet wt 5 hr)
Control Iodoacetate (5 mM) + CN (2 rnx) Hg2+ iO-5 M Cd2+ iO-5 M cl?+ 10-5 M La3+ 2 mzil Cal+ @1 miw 5rnM 10 miw A 23187 lO+ M Prostaglandin E, 10-S M Threonine 10m4 x Lysine 10-O M Histidine 10T4 M Glutamine lo-” M Cystine 10m4 M Cyst&e 10m4 M PCMPS 1O-4 x
Experimental
P for difference
42fl.2 95fl.l 5.2+ 1.2 5.9 + 0.9 6.3f0.6 62 + 0.2 6.5 & 0.6 54f@4 3.8fO.4
19.2k5.5 7.6f@6 55&0.8 7.7 + 1.1 65f06 11.6+ 1.3 3.5 * 0.3 20 * 0.4 19.2+ 1.1
< 0.05 n.s. n.s. n.s. n.s. < 0.01 < 0001 < 0001 < 0.001
59+e5 6.4 * @3 7.5 k 0.6 7.8+ 1.2 8.6 + 0.9 4,3+ 1.2 5.4kO.5 6.7 +O.Y
56f04 7.3f0‘9 6.9+ 1.6 9.5 * 1.5 8.5k1.3 8,8* 1.4 54kO.3 6.6 +_ 0.5
n.s. n.s. n.s. n.s. n.s. < 0.05 n.s. LS.
n.s. = not statistically significant (P > 0.05). Results are as means k S.E. of six pairs of lenses.
When the external Ca concentration was reduced to 10m4 accumulation of Zn was nearly doubled, while 10 mM Ca reduced it to about one-third of the control level (Table V). The calcium ionophore A 23187 produced a remarkable increase in Zn accumulation. In the rat intestine it has been suggested that prostaglandin E, can increase transport of Zn (Song and Adham, 1978). However, we could not show an effect of this autaeoid on Zn accumulation by the rabbit lens (Table V). Zinc can bind to several amino acids, including histidine, glutamine, threonine,
ZN
METABOLISM
OF
BO3
LER’S
lysine and cystine (Prasad and Oberleas, 1970). These amino acids have been identified in the aqueous humor of rabbits (Reddy, Rosenberg and Kinsey, 1961) but only cystine had any effect on the accumulation of Zn by the lenses; an increase was observed (Table V). The reduced form of cystine, cysteine, had no effect. As cystine is a disulfide compound which could interact with membrane sulfhydryl groups we tested the effects of PCMPS which can bind such moieties at the cell surface. However. it had no effect on the accumulation of Zn. Ejlux
of Zn from the lens
Lenses were incubated for 3 hr in a medium containing 10e5 M Zn and 65Zn. They were then removed from this solution and transferred through a series of incubation media in which no Zn was present (see Methods). The loss of the accumulated Zn was measured and expressed as percentage of total initial ‘ counts ’ or in ,umoles of Zn (based on the specific activity of the Zn in the bathing media containing the s5Zn). The isotope lost in the first 10 set rinse was not however used in the calculations as this mainly represented the excess incubation media on the lens surface, though also probably some of that which is present in the extracellular space. The rate of loss of the Zn Zn remained declined progressively and after 2.5 hr about 40 “/” of the accumulated in the tissue (Fig. 1). The decline in the rate of efflux of Zn was much greater than that of the calculated decrease in concentration of the accumulated Zn in the lens (see Discussion).
A
0
150 Time
(mid
FIG. 1. Efflux of Zn from the rabbit lens. Each point (O--O) 2 my lanthanum, (o-0) no lanthanum. For The procedure is described in the Methods and Results.
represents the meanCS.E. of six further data on these experiments
measurements see Table
VI.
In view of the previous experiments suggesting that Zn may move through similar pathways to calcium we measured the effects of lanthanum on the efflux of Zn. (La can block Ca-channels, both influx and efflux, in cell membranes and inhibits the activity of the Ca pump, see Weiss (1974).) The La resulted in a decline in the rate of efflux of Zn from the lens (Table VI) as also reflected by a reduction in the percentage loss of ‘counts’ by the tissue (Fig. 1). I !I EICR3x
I’. .I. I~EXTLEY TABLE
ET
dL.
\‘I
The rate of e$lux of Zn (using 65Zn as a, tracer) frowl rabbit lenses followi~ag for 3 hr in media contuinirbg IOPM %n* Zn efflux Time
period (min)
&l 5-10 20-30 6G-90
pnol
control
c:! m41 La
0.69 + 0.08 0~13~0014 004 & 0905 0010f00017
048fti0.5 0.09 + 0908 0.024 * @002 0.006 k OGOO7
kg-’
loading
wet wt min
P for difference < < < <
0.05 0.05 0.01 00.5
C’alculated cont. of 65Zn in lens PM 10 7 .M 44
As little of the Zn normally present in the lens was exchangeable the specific activity of the Zn was assumed to be the same as in the external ‘loading’ solution. * The external media for the efflux experiment contained no added Zn. For further details see Methods and Fig. 1, Results are as means f SE. for six experiments.
As changes in the external concentration of Ca (last section) have been shown to influence the accumulation of Zn by the lens we also measured the effects of this alkaline earth metal on the efflux of Zn. Lenses were loaded with Zn by incubating them for 3 hr in a solution of lop5 M Zn, containing 65Zn. The efflux was measured as described previously. The percentage counts which remained in the lens after 30 min were 38*6+ 15 (6) in control preparations (1.8 mM Ca) and 49.2 k2.4 in paired lenses exposed to a reduced, @1 mM, concentration of Ca (P < @Ol for the difference). Such a difference persisted throughout the 150 min of the efIlux measurement. When the concentration of the Ca was increased to 10 mM in the experimental medium no significant difference was observed ; for instance after 30 min the percentage remaining in the lens was 44.4k3.5 (6), while it was 37.4+ 2.3 in the control medium. 4. Discussion The rabbit lens contains about 100 ymol kg-’ wet wt of Zn, which is double that present in the amphibian lens (Baldwin and Bentley, 1980). This Zn appears to be distributed quite uniformly throughout the organ but, as also seen in the amphibian lens, less than 10 y. of the total is readily exchangeable. Most Zn in the lens is probably incorporated with the structural components of the tissue, especially proteins and nucleic acids. The aqueous and vitreous humors contain Zn at a concentration of 10e5 M, which is only one-fourth the concentration in the blood serum. Zinc is bound to plasma proteins (Prasad, 1979) and these may restrict its movement across the ciliary body into the aqueous humor. Zinc can slowly accumulate in the lens from solutions in which the concentration is similar to that in the rabbit’s vitreous and aqueous humors (10M5 M). The accumulation is far greater than that which can be accounted for by that present in the extracellular fluids of the lens, and it can be facilitated when integrity of the tissue is compromised by the presence of metabolic inhibitors. The restrictive capacity of the cell membrane is probably compromised in the latter circumstances, but there could also be an unmasking of chemical moieties to which the metal may be able to bind. Our estimate of the exchangeable fraction of the total Zn present in the lens suggests that the ‘free’ diffusible concentration in the tissue could be about 10m5 M
ZK
METABOLISM
;io5
Oh’ LENS
or less. The concentration in the rabbit ocular fluid is also 10e5 M suggesting, when the favorable gradient of electrical potential into the cells is taken into consideration, that Zn may move down an electrochemical gradient into the intracellular fluids. There is little information about the process of Zn entry into cells (see Underwood, 1977). The available information is derived principally from studies of its absorption across the intestine and its excretion in the urine (see Underwood, 1977; Foster, Aamodt, Henkin & Berman. 1979). This process could involve combination with ligands such as amino acids (Foster et al., 1979), and prostaglandin E, (Song and Adham, 1978). Zinc accumulation by the lens was not affected by such agents with the exception of cystine, which promoted accumulation. It is possible that the latter reflects binding of the Zn to the amino acid and its subsequent uptake by the lens. The uptake of Zn can also be inhibited in tissues by the presence of other metals such as copper and cadmium (see Underwood, 1977; Kingsley and Frazier, 1979). but such an effect was not seen in the present experiments on the rabbit lens. In the amphibian lens (Baldwin and Bentley, 1980) Cd decreased accumulation of Zn while Hg increased it. The pathway which Zn may follow into the lenticular cells is unknown, but it was of special interest to observe that the accumulation by the lens was enhanced when the external Ca concentration was reduced. Uptake was also increased in the presence of the Ca ionophore A 23 187. The efflux of Ca from cells can be inhibited by lanthanum, and this effect has also been observed in the rabbit lens (Hightower, Leverenz and Reddy, 1980; McGahan et al., 1983). The efflux of Zn from the rabbit lens was also reduced by La. However, the accumulation of Zn by the rabbit lens was not changed in the presence of external La suggesting that like Ca in other tissues (see Weiss, 1974) both influx and efflux of the metal may be inhibited. In the amphibian l.ens, only the influx of Zn appeared to be blocked (Baldwin and Bentley, 1980). It is thus attractive to consider the possibility that Zn and Ca follow similar pathways in and out of the lenticular cells. These sites could be diffusion channels and even involve the activity of an active pump mechanism such as a ‘Ca pump’. In the instance of Ca both types of process are known to be inhibited by La (see Weiss, 1974). Zinc is known to antagonize the effects of calcium and they may compete for similar binding sites (Ciofalo and Thomas, 1965; Prasad, 1979). The observed inhibitory effect of low external Ca concentrations on the efflux of Zn from the lens would be consistent with a reduced exchange of external Ca for bound Zn. The rate of efflux of accumulated Zn from the lens declined progressively and could not be readily related to the concentration of the metal in the tissue, if it is assumed to be present ‘free’ in solution. This inconsistency may reflect binding of the metal to tissue components, the presence of an ‘active pump’ mechanism, and/or interference due t’o the layered structural arrangement of the lens cells. Interference to solute movements due to the latter has been noted by Schultz and Curran (1970). The preceding observations on the Zn content of the rabbits’ lens and ocular fluids, and the behavior of the metal under in vitro conditions can be used to suggest a possible pattern of Zn metabolism in the lens in viva. The concentration of Zn in the aqueous and vitreous humors is adequate to assure access of the metal into the peripheral lens cells. The Zn can enter the lens across its anterior epithelial surface (possible uptake from the posterior side has not been excluded) and passes across the plasma membrane into the cells. This process is normally somewhat restricted and may involve specific pathways which may be synonymous with Ca channels and Ca-binding sites. The amino acids present in the ocular fluids probably do not interfere with this 1%”
506
P. .I. 13EKTI,E\’
ET
AI,.
process. with the possible exception of’ cystine, which could facilitatr uptake of the met,al. Once inside the cells Zn may be bound, incorporated into tissue components or remain relatively free to move out of the lens, possibly along the same pathways by which it entered the tissue. or through an active ‘pump’ mechanism. The rate of Zn accumulation by the lens may principally reflect it,s concentration in t.he ocular fluids: which is much less than that in the plasma. The ultimate control of the Zn metabolism of the lens may be due to the processes that, secrete the ocular fluids, especially the activity of the ciliary body. There
is little
specific
information
about
the
possible
role
of Zn in maintaining
lens
t’ransparency. It has been observed that senile human cataractous lenses have low levels of Zn (Swanson and Truesdale. 1971). A Zn-deficient diet has also been shown to result in cataracts in growing Rainbow trout (Ketola, 1979). Zinc is an essential trace element, but whether or not it has a special role in the maintenance of lens transparency is unknown. However. it could be involved in the stabilization of the structure of lens proteins and the removal of toxic free radicals from the tissue (Editorial, 1978). ACKNOWLEDGMENTS This work was supported by National We are grateful to Mr John Morris for
Institutes technical
of Health assistance.
grants
EY 01278
and EY 04755.
REFERENCES Baldwin, G. A. and Bentley, P. J. (1980). The zinc metabolism of the amphibian lens. Exp. Eye Res. 30, 333-43. Burnet. F. M. (1981). A possible role of zinc in the pathology of dementia. Lancet i, 18&8. Chvapil, M., Elias. S. L.. Ryan, J. N. and Zukoski, C. F. (1972). Pathophysiology of zinc. International Review of Neurobiology, Suppl. 1 (Ed. Pfeiffer, C. C.). Pp. 15-24. Academic Press, New York and London. Ciofalo, F. R. and Thomas, L. J. (1965). The effects of zinc on contractility, membrane potentials, and cation content of rat atria. J. Gen. Physiol. 48, 825-39. Editorial (1978). A radical approach to zinc. Lancet i, 191-2. Foster, D. M.. Aamodt, R. L., Henkin. R. I. and Berman, M. (1979). Zinc metabolism in humans: a kinetic model. Am. J. Physiol. 237, R34&9. Galin. M. A., Nano, H. D. and Hall. T. (1962). Ocular zinc concentration. Invest. OphthaZmoZ. 1. 142-8. Hightower, K. It., Leverenz, V. and Reddy. V. N. (1980). Calcium transport in the lens. Invest. Ophthalmol. Vis. Sci. 19, 105%66. Ketola, H. G. (1979). Influence of dietary zinc on cataracts in rainbow trout. J. Nutr. 109, 96.59. Kingsley, B. S. and Frazier, J. M. (1979). Cadmium transport in isolated perfused rat liver: zinc-cadmium competition. Am. d. Physiol. 236. C 13S43. McGahan, M. C., Chin, B. and Bentley. P. .J. (1983). Calcium metabolism of the rabbit lens. Exp. Eye Res. 37, 57-66. Prasad, A. S. (1979). Clinical, biochemical, and pharmacological role of zinc. Attn. Rut’. Pharmacol. Toxicol. 20, 393426. Prasad. A. S. and Oberleas, D. (1970). Binding of zinr to amino acids and serum proteins in vitro. J. Lab. C&n. Med. 76. 416-25. V. E. (1961). Steady state distribution of free Reddy, D. V. N., Rosenberg, C. and Kinsey. amino acids in the aqueous humours. vitreous body and plasma of the rabbit. Exp. Eye Res. 1, 175-81. Schultz, S. G. and Curran, P. F. (1970). Coupled transport of sodium and organic solutes. Physiol. Rev. 50, 637-718. Song, M. K. and Adham, N. F. (1978). Role of prostaglandin E, in zinc absorption in the rat. Am. J. Physiol. 234, E94-105.
ZS
METABOLISM
OF
LESS
507
Swanson, A. A. and Truesdale, A. W. (1971). Elemental analysis in normal and cataractous human lens tissue. Biochem. Biophys. Res. Commun. 45, 1488-96. Ulmer. D. D. (1977). Trace elements. New Engl. J. Med. 297, 318-21. Underwood, E. J. (1977). Trace elements in human and animal nutrition. fourth edition. Pp. 19&242. Academic Press, New York. San Francisco, London. Weiss, G. B. (1974). Cellular pharmacology of lanthanum. ,4nn. Rev. Pharmacol. 14. 343-5-C.