Investigative Ophthalmology & Visual Science, Vol. 30, No. 5, May 1989
Copyright © Association for Research in Vision and Ophthalmology
Pyridine Nucleotides ond Phosphorylation Potential of
Rabbit Corneal Epithelium and Endothelium
Barry R. Masters,* Amal K. Ghosh,f Jeanne Wilson,f and Franz M. Marschinskyf
In order to validate in situ corneal redoxfluorometry,the redox state and phosphorylation potential of
freeze trapped rabbit corneal epithelium and endothelium were studied using quantitative histochemical methods. The results were compared with noninvasive measurements using an optically sectioning
fluorometer microscope. Enucleated rabbit eyes were either frozen in Freon-12, cooled by liquid
nitrogen or exposed for 1 hr in 1 mM NaCN to block oxidation and then freeze trapped. Corneas were
sectioned, freeze-dried, samples of individual layers dissected, weighed, and analyzed for: NADH,
NAD + , NADPH, NADP + , ATP, ADP, and P,. The aerobic epithelium showed a ratio for NAD + /
NADH of 1.85 ± 0.08 (9). In anoxia this ratio decreased to 1.06 ± 0.07 (8). The NAD/NADH ratio
of aerobic endothelium was 3.25 ± 0.28 (6); in anoxia this ratio was 0.68 ± 0.14 (5). The values of
phosphorylation potential ATP/(ADP X P,) M ' were: 447.9 ± 40.2 (9) in aerobic epithelium, 378.2
± 24.7 (5) in anoxic epithelium; 308.4 ± 25.2 (7) in aerobic endothelium and 225.4 ± 19.1 (5) in anoxic
endothelium. Aerobic-anoxic transitions alter the concentrations of NADH and NAD + but did not
affect the concentration of NADPH and NADP + . The microhistochemical data indicate that the redox
state of rabbit epithelium is less sensitive to hypoxia than the endothelium. This difference between
the two limiting layers is reflected in alterations of phosphorylation potential induced by hypoxia. The
similarly high efficiencies of both layers in maintaining relatively high ATP levels during histotoxic
hypoxia is most likely a result of compensatory ATP generation by enhanced glycolysis. Invest
Ophthalmol Vis Sci 30:861-868,1989
The pyridine nucleotides are cofactors which interlink many metabolic reactions. The pyridine nucleotide redox ratios (NAD+/NADH and NADP + /
NADPH) are determined by the oxidizing and reducing chemical reactions in the cornea.1 Cyanide is
a reversible respiratory inhibitor which binds to the
ferric form of cytochrome oxidase in the mitochondrial respiratory chain, and prevents the generation of
ATP by oxidative phosphorylation. Cyanide can also
bind to ferric and cupric forms of other enzymes.
Since NADH is continuously synthesized and there is
no functioning pathway to oxidize it in the presence
of cyanide, the concentration of NADH increases. A
decrease in the availability of oxygen leads to a similar result. A second oxidative pathway in the cornea is
the hexose-monophosphate shunt which converts
glucose to pentose and leads to the reduction of
NADP+ to NADPH. NADPH is required for glutathione-coupled redox reactions providing protection
against oxidative damage of corneal cells. The active
transport systems in the limiting layers of the cornea
are dependent upon metabolic energy generated by
glycolysis and by oxidative phosphorylation. Glycolysis and respiration maintain the phosphorylation
potential, 23 which is denned as ATP/(ADP X Pj)
M" 1 . The phosphorylation potential is the molar
concentration of ATP divided by the product of ADP
and Pj (inorganic phosphate) and is directly related to
the free energy available from ATP.
Redox fluorometry4"6 is a noninvasive optical
method for estimating the amount of reduced pyridine nucleotide in the tissue. The fluorescence emission obtained in the region of 450 nm in response to
excitation wavelength at 366 nm contains components from reduced pyridine nucleotides (NADH and
NADPH) present in both the cytoplasm and the mitochondria, and from nonspecific fluorophores. The
bound pyridine nucleotides in the mitochondria have
a higher quantum yield, which precludes direct calibration of the analytical method. The main advantage of the fluorometric redox technique is that it can
be applied in situ, and therefore provide the basis of a
From the Departments of 'Ophthalmology, Emory University,
Atlanta, Georgia, and fBiochemistry and Biophysics, University of
Pennsylvania, Philadelphia, Pennsylvania.
Presented in part at the Annual Meeting of the Association for
Research in Vision and Ophthalmology, Sarasota, Florida, May
4-8, 1987.
Supported by National Institutes of Health Grant EY-06958
AM 19525, and Research to Prevent Blindness, Inc.
Submitted for publication: July 25, 1988; accepted November
21,1988.
Reprint requests: Barry R. Masters, PhD Emory University
School of Medicine, 1327 Clifton Road, Atlanta, GA 30322.
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INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / May 1989
clinical noninvasive diagnostic technique7 to evaluate
cellular respiratory function. Quantitative microhistochemical analysis of the pyridine nucleotides has
the advantage of measuring directly and quantitatively both the reduced and the oxidized pyridine nucleotides and related metabolites and cofactors of energy metabolism; however, the technique is destructive.
The purpose of the current study is to compare the
fluorometric redox technique with the quantitative
microhistochemical analysis, and to relate hypoxic
alterations of redox state and phosphorylation potential of the corneal epithelium and endothelium. Furthermore, the possible difference in the sensitivity between the epithelium and endothelium to acute hypoxia was investigated. The study of cyanide-inhibited
cytochrome oxidase represents complete hypoxia,
while the aerobic cornea represents the other endpoint. Further studies involving the long-term effects
of chronic hypoxia may yield different conclusions
due to adaptive mechanisms such as alterations of
enzyme activity and metabolite concentrations.
Materials and Methods
Microhistochemical Analysis8'1'
Tissue preparation: The studies were carried out
using male New Zealand white rabbits weighing
2.5-3.0 kg. The rabbits were maintained and handled
in accordance with the ARVO Resolution on the Use
of Animals in Research. The rabbits were anesthetized with an intramuscular injection of ketamine
HC1, (40 mg/kg) and xylazine, (5 mg/kg). Lidocaine
HC1 (20 mg/ml; 0.8 ml/eye) was given as a retrobulbar injection. The eyes were freed of adhering tissue,
swiftly enucleated, and frozen within 3 sec in Freon12 cooled to -150 c C. with liquid nitrogen. The frozen tissue was stored at —80°C.
Histotoxic anoxia was induced by suspending the
eyes for 1 hr in a physiological saline solution (0.9%
NaCl) containing sodium cyanide (1 mM) and
HEPES (20 mM) adjusted to pH 7.0 at 25°C. The
cyanide was weighed and the solution prepared immediately prior to the enucleation.
Tangential sections (10 ixm) were cut from the corneas using a cryostat cooled to -20°C. The sections
were then freeze-dried for 24 hr at -40°C. The epithelium, endothelium, and stromal sections were microdissected from the surrounding tissue and weighed
on a quartz fiber balance under magnification. The
sample weights varied between 0.05 and 0.1 /*g with
the endothelium usually requiring pooling of two to
three samples to reach the designated weight range.
The tissue was stored in the wells of oil well racks
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Vol. 30
under vacuum at -20°C. until the assay was performed.
Chemical analytical procedures: ATP, ADP, Pj (inorganic phosphate), NADH, NAD + + NADH,
NADP+ + NADPH, were analyzed by oil well techniques. Reduced and total nucleotides were determined and the oxidized nucleotides were calculated
by the difference between these two quantities. Enzyme cycling methods were used for NAD(H) and
NADP(H).8"10 Assays were conducted following published procedures." The concentrations (or content)
of pyridine and adenine nucleotides are expressed in
units of millimoles per kilogram of dry weight to ensure consistency between different tissues measured
in different laboratories.
Fluorometric Redox Analysis4712
The redox state of rabbit corneas was determined
prior to and after incubation with cyanide (to induce
histotoxic hypoxia) using an optically sectioning fluorometer microscope.12 The cornea was excited at an
excitation wavelength of 366 ± 10 nm and the fluorescence is measured at an emission wavelength of
450 ± 10 nm using the optically sectioning corneal
fluorometer microscope. The redox fluorometer microscope was used to measure the intensity of the
pyridine nucleotide fluorescence from the corneal epithelium and endothelium. Immediately following
enucleation, the intensity profile along the optic axis
was determined. The eye was then incubated in the
cyanide solution for 1 hr, after which the intensity
profile was again determined (Fig. 1). The difference
between the two intensity profiles was taken as a
measure of the altered degree of reduction of the pyridine nucleotides of the component layers of the cornea. The percent increase of the fluorescence intensity was calculated as: 100 X (final intensity — initial
intensity)/(initial intensity).
Statistical Methods13
The data analysis was performed with RS/1 data
analysis software. The left and right eye of each rabbit
used in the study received identical treatment prior to
freezing. The set of two eyes were either frozen immediately following enucleation (labeled aerobic) or
were treated with cyanide (labeled anoxic). The significance of the difference between the aerobic and
the anoxic eyes were first analyzed by the F-test for
equality of variance. The case with equal variances
used a pooled variance t-test, and the case with unequal variances used an unpooled variance t-test. In
addition, in case the data were not normally distributed, a nonparametric test was employed (Wilcoxon
No. 5
863
ANALYSIS OF CORNEAL NUCLEOTIDE CONTENT / Masrers er al
rank sum test). All data points greater than three
standard deviations from the mean were excluded.
EPITHELIAL
SIDE
Results
The experimental results14 from both analytical
microhistochemistry and redox fluorometry analysis
of rabbit corneal epithelium and endothelium are
summarized in Tables 1-3 and Figure 2.
Pyridine Nucleotides
The concentrations and redox ratios of the pyridine nucleotides under aerobic and anoxic conditions
are shown in Tables 1 and 2 and Figure 2. The aerobic epithelium and endothelium contain two redox
couples NAD+/NADH (I) and NADP+/NADPH (II);
the former is predominantly oxidized and the latter
predominantly reduced. The epithelium contains a
total concentration of redox couple (I) components at
16 times the total concentration of couple (II). This
ratio is only eight in the endothelium. The aerobicanoxic transition for both the epithelium and the endothelium resulted in significant increases in NADH
concentration, and significant decreases in NAD +
concentration. In addition, there was a 12% statistically significant loss of total (NAD+ + NADH) following anoxia in both epithelium and endothelium.
The apparent small changes in concentrations of
NADP + and NADPH following hypoxia were not
statistically significant. The redox state of the endothelium was more oxidized than that of the epithelium, using the NAD + /NADH couple as a redox indicator. The pyridine nucleotides significantly decreased following anoxia in both the epithelium and
the endothelium. The epithelium contained NADH
at about nine times the concentration of NADPH,
and this ratio was constant for aerobic and hypoxic
epithelium. However, the aerobic endothelium only
contains NADH at 2.5 times the concentration of
NADPH, and this ratio increased to 5 in the anoxic
endothelium.
Stromal samples were also analyzed for NADH,
NAD + , and total NADH plus NAD+. The concentrations were 0.040 ± 0.060, 0.060 ± 0.004, and 0.100
± 0.004 mmol/kg dry tissue, respectively. The concentration (expressed on a dry weight basis) of
NADH in the stroma was about 2% of the concentration found in the epithelium.
Adenine Nucleotides
The concentrations and ratios of the adenine nucleotides under aerobic and anoxic conditions are
shown in Table 3. The aerobic ratio of ATP/ADP is
about 9 for the epithelium and about 6 for the endo-
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ENDOTHELIAL
SIDE
0
100
200
300
400
500
DISTANCE (MICRONS)
Fig. 1. Effect of hypoxia on the fluorescence intensity profile
through an enucleated rabbit cornea. The fluorescence was excited
with light at 366 nm and the fluorescence emission was detected at
450 nm. The lower curve corresponds to the aerobic cornea and the
upper curve corresponds to the hypoxic cornea, as described in
Materials and Methods. The peaks on the left side of the figure
correspond to the epithelium and the peaks on the right side correspond to the endothelium.
thelium. The concentrations of ATP and Pj are approximately equal in these two layers. The phosphorylation potential for aerobic epithelium is about 30%
greater than that for the aerobic endothelium. The
transition from aerobic to anoxic condition results in
significant decreases for the epithelial ATP/ADP
ratio, and for both the endothelial ATP/ADP ratio
and the endothelial phosphorylation potential. The
small apparent change of the epithelial phosphoryla-
864
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / May 1989
Table 1. Effect of hypoxia on the concentration of pyridine nucleotides of rabbit epithelium
Parameter
NADH
NAD+
Total
NAD+/NADH
% Oxidized*
% Reduced*
NADPH
NADP+
Total
NADP7NADPH
% Oxidized:):
% Reduced*
Normoxic
(mmole/kg dry wt.)
Anoxic
(mmole/kg dry wt.)
1.84 ±0.09 (9)*
3.37 ±0.14(9)
5.21 ±0.20(9)
2.14 + 0.10(8)
2.25 ±0.16 (8)
4.39 ±0.21 (8)
1.85 ±0.08 (9)
64.70 ±1.01 (9)
35.30 ± 1.01 (9)
1.06 ±0.07 (8)
51.10 ± 1.74(8)
48.90 ± 1.74(8)
-0.79 ±0.1 If
-13.60 ±2.01f
13.60 ±2.0If
0.21 ±0.01(9)
0.11 ±0.01(9)
0.32 ± 0.01 (9)
0.25 ± 0.02 (5)
0.04 ±0.01 (5)
0.29 ±0.01 (5)
0.04 ± 0.03
-0.07 ± 0.01
-0.03 ± 0.02
0.52 ± 0.07 (9)
33.20 ±3.19 (6)
66.80 ±3.19 (6)
0.14 ±0.02 (5)
12.20 ±0.94 (5)
87.80 ± 7.55 (5)
-0.38 ± 0.07t
-21.00 ±3.33t
21.00 ± 8.19t
* Numbers expressed as means ± SEM. Number of eyes given in parentheses.
tion potential was not statistically significant. Adenine nucleotides were not determined in the stroma.
Difference
(mmole/kg dry wt.)
0.30 ± 0.13t
— 1.12 ±0.21f
-0.82 ± 0.29f
t Differed significantly, P < 0.05.
t % Oxidized (reduced) is oxidized (reduced) form divided by total.
Discussion
Redox Fluorometry
Comparison of this Work with Previous
Results of Others
The effect of anoxia on the intensity of the fluorescence of the corneal epithelium and endothelium is
shown in Figure 1. The larger peak on the left represents the corneal epithelium and the small peak on
the right represents the corneal endothelium. The
lower trace is the fluorescence scan through the aerobic cornea and the upper trace is the scan through the
anoxic cornea. This figure was obtained from eye
LI 5, and is typical of the fluorescence traces recorded
in this study. The percent change of epithelial fluorescence was + 153% and the percent change of endothelial fluorescence was +48%. There was no significant
change of the fluorescence intensity from the stromal
region.
In order to compare the chemical analytical results
of this work with those of previous work, it is necessary to convert the units15 of concentration. The normal rabbit corneal hydration is 77%. There are 3.4 g
of water/g dry weight of tissue. The following calculation was made: (concentration/kg wet wt) X 4.4
equals (concentration/kg dry wt).
Morley and Toth16 measured the pyridine nucleotides of scraped rabbit epithelium. They reported
values as: 4.22 mmol/kg dry wt for (NAD +
+ NADP+) and 2.8 mmol/kg dry wt for (NADH
+ NADPH). These values are somewhat higher than
those reported in the current study for the epithelium.
Table 2. Effect of hypoxia on the concentration of pyridine nucleotides of rabbit endothelium
Parameter
NADH
NAD+
Total
NAD7NADH
% Oxidized*
% Reduced*
NADPH
NADP+
Total
NADP7NADPH
% Oxidized*
% Reduced*
Normoxic
(mmol/kg dry wt.)
Anoxic
(mmol/kg dry wt.)
Difference
(mmole/kg dry wt.)
0.48 ± 0.04 (6)*
1.58 ±0.18 (6)
2.06 ±0.21 (6)
1.12 ± 0.18 (5)
0.74 ±0.17 (5)
1.86 ±0.28 (5)
0.64±0.18t
-0.84 ± 0.24t
-0.20 ± O.35t
3.25 ± 0.28 (6)
76.00 ± 1.53(6)
24.00+ 1.53(6)
0.68 ±0.14 (5)
38.90 ±4.78 (5)
61.10 ±4.78 (5)
-2.57 ± 0 . 3 I t
-37.1O±5.02t
37.10+ 5.02t
0.18 ±0.02 (6)
0.06 ±0.01 (6)
0.24 ± 0.03 (6)
0.23 ± 0.02 (5)
0.04 ± 0.02 (5)
0.27 ± 0.03 (5)
0.35 ± 0.02 (6)
26.00 ± 1.29(6)
74.00+ 1.29(6)
0.17 ±0.06(5)
13.80 ±4.30 (5)
86.20 ± 4.30 (5)
• Numbers expressed as means ± SEM. Number of eyes given in parentheses.
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0.05 + 0.03
0.02 ± 0.02
0.03 ± 0.04
-O.18 + O.O6t
- 1 2 . 2 0 + 4.49t
12.20 ± 4 . 4 9 t
t Differed significantly, P < 0.05.
$ % Oxidized (reduced) is oxidized (reduced) form divided by total.
ANALYSIS OF CORNEAL NUCLEOTIDE CONTENT / Mosrers er al
No. 5
865
Table 3. Effect of hypoxia on the adenine nucleotides and phosphorylation potential
of rabbit corneal epithelium and endothelium
Parameter
Anoxic
(mmol/kg dry wt.)
Normoxic
(mmol/kg dry wt.)
Difference
(mmol/kg dry wt.)
Epithelium
ATP
ADP
Pi
ATP/ADP
Phosphorylation
potential^
21.07 ±
2.31 ±
21.33 ±
9.34 ±
0.52(9)*
0.12(9)
0.94(9)
0.59(9)
19.66 ±
2.75 ±
19.13 ±
7.17 ±
447.90 ± 40.20 (9)
0.72(5)
0.99(5)
0.84(5)
0.30(5)
-1.41 ±
0.44 ±
-2.20 ±
-2.17 ±
378.25 ± 24.76 (5)
0.80
l.OOf
1.26
0.67t
-69.65 ±47.21
Endothelium
ATP
ADP
Pi
ATP/ADP
Phosphorylation
potential^
17.73 ±
2.91 ±
20.48 ±
6.26 ±
0.64(7)
0.18(7)
0.91 (7)
0.48(7)
16.86 ±
3.37 ±
22.57+
5.02 ±
308.46 ± 25.22 (7)
0.59(5)
0.09(5)
1.05(5)
0.26(5)
-0.88 ±
0.46 ±
2.09 ±
-1.24 ±
225.40 ± 19.14(5)
• Numbers expressed as means ± SEM. Number of eyes given in parentheses.
0.87
0.20
1.39
0.54f
-83.05 ± 3 1 . 6 5 t
t Differed significantly, P < 0.05.
% Phosphorylation potential expressed as ATP/(ADP X Pj) M" 1 .
A previous study by Masters et al17 on the pyridine
nucleotide levels of rabbit epithelium and endothelium differed from the current study in two respects:
azide was used to induce histotoxic anoxia, and the
samples were obtained by scraping the tissue without
freeze-trapping. The use of freeze-trapped material is
thought to result in smaller losses of total pyridine
nucleotides following anoxia. The process of scraping
the tissue may alter the tissue metabolite levels. This
could have led to disparate results between the two
methods.
Another study of the pyridine nucleotides of rabbit
corneal epithelium used the cycling assay on tissue
samples that were not freeze-trapped.18 The only result with the same value is the NAD + /NADH redox
ratio for aerobic epithelium. The outcome for all
other determinations differed.
Reim et al'9"22 reported nucleotide concentrations
and redox ratios on freeze trapped tissues. For bovine
aerobic epithelium, he measured NADP+/NADPH
ratio of 0.70 compared with 0.52 measured in this
study. The measured ATP/ADP ratios for epithelium
NADH
NAD
NADH + NAD
Fig. 2. The concentrations of pyridine nucleotides under conditions of
normoxia and hypoxia in
rabbit epithelium and endothelium. In all Figures, the
error bars show standard
errors of the mean.
NAD + /NADH
•
NADPH
EPINORMOXIC
H EPI-ANOXIC
+
M ENDONORMOXIC
NADP
NADPH + NADP
H ENDOAN0XIC
+
NADP /NADPH
I I I i i l l I I I
1
2
3
4
MMOL/KG DRY WT OR RATIO
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l I I I I
5
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / May 1989
(12.8) and endothelium (6.9) are very close to the
values reported in the current study. The phosphorylation potential of aerobic toad corneal epithelium23
obtained by scraping was measured to be 599 ± 226
NT1. These independent investigations indicate the
reliability of freeze-trapping, and also indicate the
great resistance of corneal epithelium to hypoxia.
Comparison of Differences between Epithelium and
the Endothelium
The microhistochemical method measures the
total cellular amount of nucleotide expressed in terms
of mmol/kg dry wt of tissues. Two levels of complexity are to be dealt with in the interpretation of the
data thus obtained. The first level is the anatomical
heterogeneity of the tissue sample. Since the samples
were usually taken from the central cornea, we can
ignore possible differences between central and peripheral regions. The analytical method measures the
average value of the cells in the sample. Therefore,
cell-to-cell differences within a sample cannot be detected. For endothelial samples, the single cellular
layer does not present a problem if it is assumed that
all the endothelial cells are in the same metabolic
state. However, if there exists heterogeneity of metabolic states,24 this assumption would not be valid.
The epithelial layers exhibit differences in anatomical
structure and chemical composition. The epithelium
consists of a basal cell layer of high mitotic and metabolic activity and four additional layers with diminishing metabolic activity in the direction of the tear
film. Desquamating epithelial cells probably have
minimal metabolic activity. Although quantitative
histochemical technology offers the spatial resolution
to differentiate between these defined epithelial
layers, that approach was not taken here for practical
reasons.25 The second level of complexity involves
the compartmentalization of metabolites in the cells.
The nucleotides exist in the mitochondrial space and
in the cytoplasmic space. They can be bound to enzymes or exist in the free state. There can be local
pools of nucleotides that are at different concentrations than the average concentration determined by
the analytical microchemical methods. The analysis
of such compartmentation is beyond the resolution
of quantitative histochemistry.
Differences between aerobic epithelium and endothelium were measured. Both the concentrations
(content reported on a dry weight basis) of NADH
and NAD"1" are higher in the epithelium. The cellular
redox ratio given by NAD+/NADH is greater in the
endothelium. Thus, the endothelium appears to be
more oxidized than the epithelium. A similar conclusion was reached by Chance and Lieberman,5 who
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Vol. 30
used fluorometry to measure the fluorescence intensity ratio of the flavoproteins2627 to the pyridine nu^
cleotides for freeze-trapped epithelium and endothelium. It should be stressed that while quantitative
histochemical techniques for pyridine nucleotides
(cytoplasmic and mitochondrial) are well developed,
equivalent quantitative histochemical techniques for
the flavoproteins do not exist. The specific flavoproteins responsible for the redox fluorometric signal
(450 nm excitation, 550 nm emission) have never
been characterized. The analytical result that the endothelium is more oxidized is also consistent with its
higher rate of oxygen uptake2829 and its aerobic enzyme activity.30-31 The results of the redox potential
measurements are consistent with the results of adenine nucleotide measurements. Both the phosphorylation potential and the ratio of ATP/ADP were significantly higher in the epithelium than in the endothelium.
These results are consistent with the rabbit corneal
epithelium being more resistant to alterations of its
redox state induced by hypoxia than the endothelium. One may postulate that this property serves to
protect the epithelium from hypoxic stress, which can
occur in cellular injury, lid closure and contact lens
wear. The effect of anoxia on the phosphorylation
potential showed similar characteristics. The epithelium was less sensitive to hypoxia than the endothelium, as indicated by the 50% smaller decrease in
phosphorylation potential.
The different concentrations of (NADH + NAD+)
and (NADPH + NADP+) between the epithelium
and the endothelium are probably related to different
levels of mitotic activity in the two layers. Reim20 has
demonstrated that NADP-dependent enzymes in the
corneal epithelium reflect high levels of synthetic activity associated with cell replication. The endothelium has significantly lower levels of synthetic activity and this is evident in the lower levels of enzyme
activity relating to synthesis. This difference in cellular function is also demonstrated by the data presented in the present paper.
The apparent difference between corneal endothelium and epithelium in sensitivity to histotoxic anoxia should, however, not be overemphasized because the energy metabolism of both tissues appears
to be designed to deal effectively with hypoxia, as
demonstrated by the maintenance of ATP levels. The
ATP/ADP ratio and the phosphorylation potential,
both sensitive indicators of the energy state of the cell,
are also remarkably stable and resistant to the effect
of 1 raM cyanide. It is not unreasonable to assume
that ATP generation by enhanced glycolysis compensates for impaired oxidative phosphorylation32 of
all corneal cell layers.
No. 5
ANALYSIS OF COP.NEAL NUCLEOTIDE CONTENT / Masters er al
Comparison of Microchemical Analysis and Redox
Fluorometry
The fluorescence intensity increased for both the
epithelium and the endothelium; however, there was
no change measured in the stromal region. Three
causes may account for this result: (1) the stromal
fluorescence is not due to reduced pyridine nucleotides but originates from nonspecific fluorescence; (2)
low levels of completely reduced pyridine nucleotides
are present in the keratocytes but anoxia has no effect
on their redox state; and (3) the keratocytes contain
the NAD + /NADH redox couple that is altered in anoxia, but the failure to measure changes of fluorescence is due to the large nonspecific fluorescence
level of the stromal region. The quantitative histochemical analysis of the stromal tissue is consistent
with the first hypothesis.
If the fluorescence was due to a homogeneously
distributed concentration of NADH, then a doubling
of the NADH concentration would result in a doubling of the fluorescence intensity. Anoxia resulted in
epithelial NADH concentration increasing 16%; the
fluorescence intensity increased 158%. The fluorescence intensity is proportional to the quantum yield.
The quantum yield of the reduced pyridine nucleotides is significantly increased3334 when it is bound to
dehydrogenase in the mitochondria, and this results
in increased fluorescence intensity. The discrepancy
between the analytical and the fluorometric results in
the anoxic transition of the epithelium is consistent
with the following argument. Redox fluorometry of
the epithelium measured the increase of the mitochondrial pool of bound (high quantum yield)
NADH. While the analytical method measured the
total increase of NADH (16%), the fraction of the
bound NADH underwent a large increase. The analytical method cannot discriminate between the
bound and the free NADH; however, the redox fluorometry is extremely sensitive to the amount of
bound NADH.
The analytical results confirm the following conclusions: under conditions of anoxia, redox fluorometry measured changes of NADH concentrations,
since the NADPH concentrations are not significantly altered in anoxia. The data are consistent with
the interpretation that the bound mitochondrial pool
of NADH might be the source of the fluorescence
changes.
Redox fluorometry has a potential resolution of 0.5
nm along the optic axis, and single-cell resolution in
the plane of the cornea.35-36 An absolute calibration of
redox cellular hypoxia can be defined that could give
a useful measure of normal and altered respiratory
function. The maximum value of fluorescence could
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867
be defined at the point of 100% hypoxia. A graded
scale of cellular hypoxia may then be constructed
from these endpoints and be used to measure cellular
respiratory function.
Key words: adenine nucleotides, cornea, phosphorylation
potential, pyridine nucleotides, redox fluorometry
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