Exp. Eye Res. (1975) 20, 393-396
The Metabolism of the Bovine Lens in Air and Nitrogen RUTH VAN HEYNINGEN
AND JANE LINKLATER
Xu$idd Laboratory of Ophthalmology, University of Oxford. Walton Street, Oxford OX.2 6AW? 1lVi. (Received8 January 19X5: Lonhl)
Anoxia had an adverse effect on the metabolism of the adult bovine lens incubated in a tissue culture medium (TC 199),buffered with HEPES and containing 1.60 mM-calcium. The incorporation of [14C]tyrosine into protein, and the concentration of ATP, were decreased. Lactate formation was considerably, and sodium concentration marginally, increased. These results are very similar to those obtained using a much simpler medium consisting of inorganic salts (Krebs-Ringer) buffered with phosphat’e and containing 1.38mni\zcalcium (Trayhurn and van Heyningen, 1972).
1. Introduction Trayhurn and van Heyningen (1972) reported that anoxia had an adverse effect on the adult bovine lens incubated in vitro, and concluded that respiration plays a substantial role in its metabolism. The concentration of calcium in the incubation nledium is an important factor in maintaining the integrity of the lens (Thoft and Kinoshita: 1965). Although calcium in the calf aqueoushumour is about 1.2 ILIM, a higher concentration (2.3 111~) was needed in vitro to maintain the normal Na-+ and Kf concentrations in the calf’ lens (Merola, Kern and Kinoshita, 1960). We measured calcium in the aqueous humour df the adult bovine eye and found a value of 146 & 0.08 mM (11 determinations & S.D.; range 1.37-1.66 mM). The medium used by Trayhurn and van Heyningen (1972) contained calcium at a concentration at the lower end of thk range (l-38 mM) but in the presence of about eight t,imesits concentration of phosIhate it may have been “bound” and partly “unavailable”. As the calcium concentration may therefore have been below the physiological level we decided to repeat the experiments replacing phosphate buffer with HEPEIC: (N-2-hydroxyethylpiperazine-IV’-2-ethanesulphonic acid) which buffers optimally in the physiological range and doesnot bind cations. Since our aim was to seeif normal lens metabolism is dependent on oxygen, we did not wish to use a concenbration of calcium outside the physiological range and therefore decided on 1.6 mM. near thtl highest value found in the bovine aqueous humour. TVe also used tissue cultural medium TC 199 in place of Krebs-Ringer. 2. Materials and Methods Most of the methods have been describedpreviously (Trayhurn and van He,vningen. 1972). TC 199 (Difco Laboratories, Detroit, Michigan, U.S.A.) which is a chemically defined
medium resembling serum ultrafiltrate, contains glucose (4-5 mM) phosphate (0436 mM) and calcium (l-26 mM). The incubation medium (100 ml) consisted of 80 ml of TC 199; 1’7ml of HEPES (82 mu)
393
394
R.
VAN
HEYNIN(:EX
Xpl’D
L.
LINKLATER
adjusted to pH 7.4 with NaHCO,; 1 ml of penicillin and streptomycin (I( 1 mg of eac*h/nll) ; 1 ml of CaCl, (59 mM) and 1 ml of glucose (O-56 M). The final concentration of gluc~~~~ in the incubation was 10 mM, calcium 1.6 mu and phosphate O-69 mu. HEPES was bought from Stuart, Kinney and Co. Ltd, 11 Argyll St., London, Wl A 4ES. Pairs of lenses weighing 2.0-2.3 g each were incubated for 22 hr at 35”C, one in air iilltl the other in nitrogen, each in 15 ml medium containing L[U-14C]tyrosine (3 @ii. In the earlier series [14C]tyrosine in the medium was added without carrier and was at it final concentration of 0.39 pmol/l. In this series unlabelled tyrosine was present in the TC 199 at a final concentration of 0.18 mmol/l. Preparation of extracts, and assay of sodium and ATP were as before. Lactate was measured in the medium using an enzymatic method (Hohorst, 1963). Radioactivity of the protein and of perchloric-acid extracts was measured on an LKB-Wallac liquid scintillation counter. Calcium was measured in bovine aqueous humour by the method of Kepner and Hercules (1963) modified by G. K. Turner Associates (24-25 Pulgas avenue, Palo Alto, California 94303, U.S.A.).
3. Results These are given in Table I, and compared TABLE
with
the earlier
series.
I.
Incubation of the bovine lens, in air and nitrogen: comparisonbetweentwo dijgPerent media Incubation
Compoaiton
medium
TC 199-HEPES (This paper)
of medium
Krebs-Ringer-Phosphate [Trayhurn and van Heyningen (1972)]* m4
(mN
Calcium Phosphate Glucose HEPES [l*C]Tyrosine (3 PCi) Amino acids and vitamins PH
1.60 0.69 10.00 15.00 0.18 Present 7.4
Incubation
1.38 11.29 10.00 -0*0004 Absent 7.4
of lens water)
I Air 17.451.5
II n’itrogen 20?5f3*9
100 x II/I 123(b)
[l*C]tyrosine in protein (Ct/min x 10e3/g of lens)
53.7A9.S
26.5f4.8
49(d)
111(a)
Naf
(pmol/g
[l*C]tyrosine in HClO,soluble material (Ct/min 1O-3/g of lens) ATP
(pmol/g
of lens)
Lactate formed (pmol/g of lens in 22 hr)
I Air 17.7fl.O
II Nitrogen 19*1*1.2
100 x II/I I OS(“) 47((l)
x 157fll
175&19
1.37f0.12
o+39fo~os
30.4&4.6
50.1&
Results are expressed as mean values & to an initial value in the medium of 5500 x P values from unpaired Students’ t tests (e) P < 0.001. * The lactate values have been corrected
65(e)
7.6
165(e)
124(d) 1.61&0.04
1*09&0*11
32.3f2.1
47.Sf2.6
68(e)
14sW
s.d. of six to eight observations. Radioactivities are adjusted lo3 ct min-l 15 ml-l. (a) not significant; (b) P < 0.1; (c) P < 0.05 ; (d) P < O-01; as a faulty
standard
was used in that
paper.
THE
BOVINE
LENS
IN
AIR
AND
NITROGEX
396
Sodium in the lens incubated in nitrogen was 20.5 ,umol/g lens water, compared with 17.4 pmol/g water after incubation in air. This is a slightly greater difference, due to anoxia, than that found when the Krebs-Ringer phosphate was used, but the standard deviations are greater and the difference barely significant. Incorporation of [14C]tyrosine into lens protein Tyrosine is not metabolized by the bovine lens (Trayhurn and van Heyningen, 1973), apart from its incorporation into protein. Incorporation was reduced by anoxia to 49% of its value in air, a figure very close to the 47% found in the earlier series. There was no significant difference in the radioactivity found in the HClO,-soluble material; in the earlier series there was a somewhat higher count in the absence of air. In the present series, where the concentration of tyrosine in the medium was about GO times higher than previously, anaerobiosis did not affect the transport of bhe amino acid into the lens.
,4 TP ATP in the lens, after incubation in the medium buffered by HEPES, was reduced to 65:/, by anoxia; the equivalent figure after incubation in the Krebs-Ringer phosphate was 68%. Since the average value for the ATP concentration in six fresh bovine lenses was 1.42 &O-l0 ,umol/g, aerobic incubation in the HEPES medium did not result in net loss of ATP. The higher value in the earlier series after incubation in air is probably because those lenses were somewhat lighter (and younger)-about :! g compared with about 2.15 g.
Lactate In nitrogen, the lactate production was increased to 165% of the aerobic value (Pasteur effect). In the earlier series the increase was to 14876. 4. Discussion The effect of anoxia on the metabolism of the adult bovine lens was very similar to that found in the earlier experiments (Table I), when the concentration of calcium in the medium was a little lower and that of phosphate much higher. There is no doubt that oxidative metabolism occurs in the lens, in the layer of epithelial cells which contain mitochondria and are in proximity to the aqueous humour. The bovine lens oxidizes the amino acids glutamate, aspartate, glutamine and asparagine with the production of CO,, * others are oxidized to a lesser extent (Trayhurn and van Heyningen, 1972, 1973). A Pasteur effect is exhibited by bovine (young and old) and rabbit lens (Kern, 1962, Table I and van Heyningen, 1965). Furthermore, in the absence of glucose, oxygen enables the bovine lens, both calf and adult, to derive a substantial amount of energy from its endogenous substrates (Kinoshita, Kern and Merola, 1961; Trayhurn and van Heyningen, 1972). There is some difference of opinion about the ability of the lens to maintain its nietabolic integrity in vitro by anaerobic glycolysis alone. The fact that more lactate is produced in nitrogen than in air shows that the lens generates more ATP by glycolysis when under anaerobic conditions. The question is, to what extent is this ATP aableto support the energy-requiring reactions of the lens? The answer seems to be that ATP production by anaerobic glycolysis is almost, or entirely, adequate to support a
396
It.
VAN
HEYNINGEN
AND
J.
LI,R;KLAr[‘E:K
variety of metabolic processes. Incorporation of tyrosine into protein Irjy the adult bovine lens is an exception; in this case lack of air is a disadvantage; tyrosinth incorporation is halved and this is accompanied by a marked decrease in concentratioil of ATP (Table 1). Examples of studies in which anaerobic glycolysis has been shown to be largely 01 entirely adequate are the recovery of cations to normal levels after exposure to cold (Harris, Gruber, Talman and Hoskinson, 1959; Kinoshita et al., 1961), the transport of amino acids into the lens (Kern, 1962; Table I and Trayhurn and van Heyningen. 1972) uptake by the lens of a-amino isobutyric acid (Kinsey and Reddy, 1963; Kinsey, 1965) of rubidium (Becker, 1962) and of inositol (Varma, Chakrapani and ReddJ-. 1970). It appears that somemetabolic processesin the lens are sensitive to lack of oxygen while others are not. Probably there are also ageand speciesdifferences and the details of the experimental conditions of incubation may also be important. ACKNOWLEDGMENTS
We thank Dr P. Trayhurn for useful discussionsand Dr J. H. Kinoshita and Dr S. D. Varma
for help in the preparation
of this paper. REFERENCES
Becker, B. (1962). Accumulation of rubidium-86 by the rabbit lens. Invest. Ophthalmol. 1, 502. Harris, J. E., Gruber, M. S., Talman, E. and Hoskinson, G. (1959). The influence of oxygen on the photodynamic action of methylene blue on cation transport in the rabbit lens. Awer. */. Ophthalmol. 48, 528. Hohorst, H. J. (1963). In Methods of Enzymatic Analysis (Ed. Bergmeyher, H. V.). 1st ed., pp. 266-70. Verlag Chemie Weinheim, Academic Press, New York. Kepner, B. L. and Hercules, D. M. (1963). The fluorimetric determination of calcium in blood serum. Anal. Chem. 35, 1238. Kern, H. L. (1962). Accumulation of amino-acids by calf lens. Invest. Ophthalmol. 1, 368. Kinoshita, J. H., Kern, H. L. and Merola, L. 0. (1961). Factors affecting the cation transport, of calf lens. Biokhinz. Biophys. Acta 47, 458. Kinsey, V. E. and Reddy, D. V. N. (1963). Studies on the crystalline lens. (X) Transport of amino acids. Invest. Ophthalmol. 2, 229. Kinsey, V. E. (1965). Amino acid transport in the lens. Invest. Ophthdmol. 4, 691. Merola, L. O., Kern, H. L. and Kinoshita, J. H. (1960). The effect of calcium on the cations of the calf lens. A.N.A. Arch. Ophthalmol. 63, 830. Thoft, R. A. and Kinoshita, J. H. (1965). Effect of calcium on rat’ lens permeability. Inwst. Ophthalmol. 4, 122. Trayhurn, P. and van Heyningen, R. (1972). The role of respiration in the energy metabolism of the bovine lens. Biochem. J. 129, 507. Trayhurn, P. and van Heyningen, R. (1973). The metabolism of amino acids in the bovine lens. Biochem. J. 136, 67. van Heyningen, R. (1965). The metabolism of glucose by the rabbit lens in the presence and absence of oxygen. Biochem. J., 96, 419. Varma, S. D., Chakrapani, B. and Reddy, V. N. (1970). Intraocular transport of myoinositol. (II) Accumulation in the rabbit lens in vitro. Invest. Ophthalmol. 9, 794.
The Metabolism of the Bovine Lens in Air and Nitrogen RUTH VAN HEYNINGEN
AND JANE LINKLATER
Xu$idd Laboratory of Ophthalmology, University of Oxford. Walton Street, Oxford OX.2 6AW? 1lVi. (Received8 January 19X5: Lonhl)
Anoxia had an adverse effect on the metabolism of the adult bovine lens incubated in a tissue culture medium (TC 199),buffered with HEPES and containing 1.60 mM-calcium. The incorporation of [14C]tyrosine into protein, and the concentration of ATP, were decreased. Lactate formation was considerably, and sodium concentration marginally, increased. These results are very similar to those obtained using a much simpler medium consisting of inorganic salts (Krebs-Ringer) buffered with phosphat’e and containing 1.38mni\zcalcium (Trayhurn and van Heyningen, 1972).
1. Introduction Trayhurn and van Heyningen (1972) reported that anoxia had an adverse effect on the adult bovine lens incubated in vitro, and concluded that respiration plays a substantial role in its metabolism. The concentration of calcium in the incubation nledium is an important factor in maintaining the integrity of the lens (Thoft and Kinoshita: 1965). Although calcium in the calf aqueoushumour is about 1.2 ILIM, a higher concentration (2.3 111~) was needed in vitro to maintain the normal Na-+ and Kf concentrations in the calf’ lens (Merola, Kern and Kinoshita, 1960). We measured calcium in the aqueous humour df the adult bovine eye and found a value of 146 & 0.08 mM (11 determinations & S.D.; range 1.37-1.66 mM). The medium used by Trayhurn and van Heyningen (1972) contained calcium at a concentration at the lower end of thk range (l-38 mM) but in the presence of about eight t,imesits concentration of phosIhate it may have been “bound” and partly “unavailable”. As the calcium concentration may therefore have been below the physiological level we decided to repeat the experiments replacing phosphate buffer with HEPEIC: (N-2-hydroxyethylpiperazine-IV’-2-ethanesulphonic acid) which buffers optimally in the physiological range and doesnot bind cations. Since our aim was to seeif normal lens metabolism is dependent on oxygen, we did not wish to use a concenbration of calcium outside the physiological range and therefore decided on 1.6 mM. near thtl highest value found in the bovine aqueous humour. TVe also used tissue cultural medium TC 199 in place of Krebs-Ringer. 2. Materials and Methods Most of the methods have been describedpreviously (Trayhurn and van He,vningen. 1972). TC 199 (Difco Laboratories, Detroit, Michigan, U.S.A.) which is a chemically defined
medium resembling serum ultrafiltrate, contains glucose (4-5 mM) phosphate (0436 mM) and calcium (l-26 mM). The incubation medium (100 ml) consisted of 80 ml of TC 199; 1’7ml of HEPES (82 mu)
393
394
R.
VAN
HEYNIN(:EX
Xpl’D
L.
LINKLATER
adjusted to pH 7.4 with NaHCO,; 1 ml of penicillin and streptomycin (I( 1 mg of eac*h/nll) ; 1 ml of CaCl, (59 mM) and 1 ml of glucose (O-56 M). The final concentration of gluc~~~~ in the incubation was 10 mM, calcium 1.6 mu and phosphate O-69 mu. HEPES was bought from Stuart, Kinney and Co. Ltd, 11 Argyll St., London, Wl A 4ES. Pairs of lenses weighing 2.0-2.3 g each were incubated for 22 hr at 35”C, one in air iilltl the other in nitrogen, each in 15 ml medium containing L[U-14C]tyrosine (3 @ii. In the earlier series [14C]tyrosine in the medium was added without carrier and was at it final concentration of 0.39 pmol/l. In this series unlabelled tyrosine was present in the TC 199 at a final concentration of 0.18 mmol/l. Preparation of extracts, and assay of sodium and ATP were as before. Lactate was measured in the medium using an enzymatic method (Hohorst, 1963). Radioactivity of the protein and of perchloric-acid extracts was measured on an LKB-Wallac liquid scintillation counter. Calcium was measured in bovine aqueous humour by the method of Kepner and Hercules (1963) modified by G. K. Turner Associates (24-25 Pulgas avenue, Palo Alto, California 94303, U.S.A.).
3. Results These are given in Table I, and compared TABLE
with
the earlier
series.
I.
Incubation of the bovine lens, in air and nitrogen: comparisonbetweentwo dijgPerent media Incubation
Compoaiton
medium
TC 199-HEPES (This paper)
of medium
Krebs-Ringer-Phosphate [Trayhurn and van Heyningen (1972)]* m4
(mN
Calcium Phosphate Glucose HEPES [l*C]Tyrosine (3 PCi) Amino acids and vitamins PH
1.60 0.69 10.00 15.00 0.18 Present 7.4
Incubation
1.38 11.29 10.00 -0*0004 Absent 7.4
of lens water)
I Air 17.451.5
II n’itrogen 20?5f3*9
100 x II/I 123(b)
[l*C]tyrosine in protein (Ct/min x 10e3/g of lens)
53.7A9.S
26.5f4.8
49(d)
111(a)
Naf
(pmol/g
[l*C]tyrosine in HClO,soluble material (Ct/min 1O-3/g of lens) ATP
(pmol/g
of lens)
Lactate formed (pmol/g of lens in 22 hr)
I Air 17.7fl.O
II Nitrogen 19*1*1.2
100 x II/I I OS(“) 47((l)
x 157fll
175&19
1.37f0.12
o+39fo~os
30.4&4.6
50.1&
Results are expressed as mean values & to an initial value in the medium of 5500 x P values from unpaired Students’ t tests (e) P < 0.001. * The lactate values have been corrected
65(e)
7.6
165(e)
124(d) 1.61&0.04
1*09&0*11
32.3f2.1
47.Sf2.6
68(e)
14sW
s.d. of six to eight observations. Radioactivities are adjusted lo3 ct min-l 15 ml-l. (a) not significant; (b) P < 0.1; (c) P < 0.05 ; (d) P < O-01; as a faulty
standard
was used in that
paper.
THE
BOVINE
LENS
IN
AIR
AND
NITROGEX
396
Sodium in the lens incubated in nitrogen was 20.5 ,umol/g lens water, compared with 17.4 pmol/g water after incubation in air. This is a slightly greater difference, due to anoxia, than that found when the Krebs-Ringer phosphate was used, but the standard deviations are greater and the difference barely significant. Incorporation of [14C]tyrosine into lens protein Tyrosine is not metabolized by the bovine lens (Trayhurn and van Heyningen, 1973), apart from its incorporation into protein. Incorporation was reduced by anoxia to 49% of its value in air, a figure very close to the 47% found in the earlier series. There was no significant difference in the radioactivity found in the HClO,-soluble material; in the earlier series there was a somewhat higher count in the absence of air. In the present series, where the concentration of tyrosine in the medium was about GO times higher than previously, anaerobiosis did not affect the transport of bhe amino acid into the lens.
,4 TP ATP in the lens, after incubation in the medium buffered by HEPES, was reduced to 65:/, by anoxia; the equivalent figure after incubation in the Krebs-Ringer phosphate was 68%. Since the average value for the ATP concentration in six fresh bovine lenses was 1.42 &O-l0 ,umol/g, aerobic incubation in the HEPES medium did not result in net loss of ATP. The higher value in the earlier series after incubation in air is probably because those lenses were somewhat lighter (and younger)-about :! g compared with about 2.15 g.
Lactate In nitrogen, the lactate production was increased to 165% of the aerobic value (Pasteur effect). In the earlier series the increase was to 14876. 4. Discussion The effect of anoxia on the metabolism of the adult bovine lens was very similar to that found in the earlier experiments (Table I), when the concentration of calcium in the medium was a little lower and that of phosphate much higher. There is no doubt that oxidative metabolism occurs in the lens, in the layer of epithelial cells which contain mitochondria and are in proximity to the aqueous humour. The bovine lens oxidizes the amino acids glutamate, aspartate, glutamine and asparagine with the production of CO,, * others are oxidized to a lesser extent (Trayhurn and van Heyningen, 1972, 1973). A Pasteur effect is exhibited by bovine (young and old) and rabbit lens (Kern, 1962, Table I and van Heyningen, 1965). Furthermore, in the absence of glucose, oxygen enables the bovine lens, both calf and adult, to derive a substantial amount of energy from its endogenous substrates (Kinoshita, Kern and Merola, 1961; Trayhurn and van Heyningen, 1972). There is some difference of opinion about the ability of the lens to maintain its nietabolic integrity in vitro by anaerobic glycolysis alone. The fact that more lactate is produced in nitrogen than in air shows that the lens generates more ATP by glycolysis when under anaerobic conditions. The question is, to what extent is this ATP aableto support the energy-requiring reactions of the lens? The answer seems to be that ATP production by anaerobic glycolysis is almost, or entirely, adequate to support a
396
It.
VAN
HEYNINGEN
AND
J.
LI,R;KLAr[‘E:K
variety of metabolic processes. Incorporation of tyrosine into protein Irjy the adult bovine lens is an exception; in this case lack of air is a disadvantage; tyrosinth incorporation is halved and this is accompanied by a marked decrease in concentratioil of ATP (Table 1). Examples of studies in which anaerobic glycolysis has been shown to be largely 01 entirely adequate are the recovery of cations to normal levels after exposure to cold (Harris, Gruber, Talman and Hoskinson, 1959; Kinoshita et al., 1961), the transport of amino acids into the lens (Kern, 1962; Table I and Trayhurn and van Heyningen. 1972) uptake by the lens of a-amino isobutyric acid (Kinsey and Reddy, 1963; Kinsey, 1965) of rubidium (Becker, 1962) and of inositol (Varma, Chakrapani and ReddJ-. 1970). It appears that somemetabolic processesin the lens are sensitive to lack of oxygen while others are not. Probably there are also ageand speciesdifferences and the details of the experimental conditions of incubation may also be important. ACKNOWLEDGMENTS
We thank Dr P. Trayhurn for useful discussionsand Dr J. H. Kinoshita and Dr S. D. Varma
for help in the preparation
of this paper. REFERENCES
Becker, B. (1962). Accumulation of rubidium-86 by the rabbit lens. Invest. Ophthalmol. 1, 502. Harris, J. E., Gruber, M. S., Talman, E. and Hoskinson, G. (1959). The influence of oxygen on the photodynamic action of methylene blue on cation transport in the rabbit lens. Awer. */. Ophthalmol. 48, 528. Hohorst, H. J. (1963). In Methods of Enzymatic Analysis (Ed. Bergmeyher, H. V.). 1st ed., pp. 266-70. Verlag Chemie Weinheim, Academic Press, New York. Kepner, B. L. and Hercules, D. M. (1963). The fluorimetric determination of calcium in blood serum. Anal. Chem. 35, 1238. Kern, H. L. (1962). Accumulation of amino-acids by calf lens. Invest. Ophthalmol. 1, 368. Kinoshita, J. H., Kern, H. L. and Merola, L. 0. (1961). Factors affecting the cation transport, of calf lens. Biokhinz. Biophys. Acta 47, 458. Kinsey, V. E. and Reddy, D. V. N. (1963). Studies on the crystalline lens. (X) Transport of amino acids. Invest. Ophthalmol. 2, 229. Kinsey, V. E. (1965). Amino acid transport in the lens. Invest. Ophthdmol. 4, 691. Merola, L. O., Kern, H. L. and Kinoshita, J. H. (1960). The effect of calcium on the cations of the calf lens. A.N.A. Arch. Ophthalmol. 63, 830. Thoft, R. A. and Kinoshita, J. H. (1965). Effect of calcium on rat’ lens permeability. Inwst. Ophthalmol. 4, 122. Trayhurn, P. and van Heyningen, R. (1972). The role of respiration in the energy metabolism of the bovine lens. Biochem. J. 129, 507. Trayhurn, P. and van Heyningen, R. (1973). The metabolism of amino acids in the bovine lens. Biochem. J. 136, 67. van Heyningen, R. (1965). The metabolism of glucose by the rabbit lens in the presence and absence of oxygen. Biochem. J., 96, 419. Varma, S. D., Chakrapani, B. and Reddy, V. N. (1970). Intraocular transport of myoinositol. (II) Accumulation in the rabbit lens in vitro. Invest. Ophthalmol. 9, 794.
