GLUCOSE METABOLISM AND
MAINTENANCE OF TRANSPARENCY IN
OCULAR TISSUE 0F RAINBOW TROUT

Thesis for the Degree of Ph. D.
MTCHIGAN STATE UNIVERSITY
LANCE E. OLSON
1969

 

1411‘;wa RY
Michigan Stat-'3
University

This is to certify that the

thesis entitled

Glucose Metabolism and
Maintenance of Transparency in
Ocular Tissue of Rainbow Trout

presented by
Lance E. Olson
has been accepted towards fulfillment

of the requirements for

_Eh_._lL__ degree in My

Rm" (1 MW»

Major professor

Date May 15, 1969

0469

 

     
  
    
  

  

 

 

 

 

 

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ABSTRACT

GLUCOSE METABOLISM AND MAINTENANCE OF
TRANSPARENCY IN OCULAR TISSUE
OF RAINBOW TROUT

BY
Lance E. Olson

Glucose metabolism in the rainbow trout lens and
cornea was investigated by the amounts of C1402 produced
during incubation of tissues with 2-C—14-pyruvate, glucose-
l-Cl4 or glucose-6-Cl4 as the labeled precursors. Tissues
were incubated in phosphate buffered saline containing a
labeled precursor for 4 hours at 13 C, and the C1402 yield
was determined by counting in a liquid scintillation
system. Tissue radioactivity was measured after solubili-
zation in Hyamine hydroxide for 18 hr at 60 C.

Glucose appears to enter the trout lens and cornea
by passive diffusion which is a different mechanism than
is postulated for similar mammalian tissues. Neither
lesioning the lens capsule nor use of specific inhibitors
of the citric acid cycle (TCA) on the lens or cornea
changes the rate of entry into the tissues. The entrance

of pyruvate into the tissue can be decreased when the

tissues were subjected to NaCN.

 

Lance E. Olson

In the tissue glucose is oxidized to CO2 by means
0f glycolysis and the TCA cycle in the lens and cornea
but more C02 is produced in the cornea.

The hexosemonophosphate (HMP) shunt also oxidized
glucose to CO2 but accounted for only a fraction of the
total CO2 production by the lens or cornea.

Glucose was oxidized primarily through energy
yielding reactions of the TCA cycle at the environmental
temperature of rainbow trout (13 C). As the temperature
rises, the HMP shunt responds in both types of tissues
with a greater increase in activity than the TCA cycle.
Viability of the tissue was judged by the maintenance of
transparency, and this increase in activity of the HMP
shunt lasted to 23 C in the cornea but persisted to 33 C

in the lens, the resPective temperatures where the tissues

become opaque.

 

GLUCOSE METABOLISM AND MAINTENANCE OF

TRANSPARENCY IN OCULAR TISSUE

OF RAINBOW TROUT
By

Lance E. Olson

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Physiology

1969

 

 

Dedicated to my wife, Jan, for her

understanding, tolerance, and sacrifice to

make this project possible.

 

 

 

   

 

 

ACKNOWLEDGMENTS

The author wishes to express his appreciation to
Dr. P. O. Fromm and Dr. J. R. Hoffert for their guidance
and patience throughout this study and their assistance
in the preparation of this dissertation.

The author is also indebted to the National
Institutes of Health for the support of this work through
grant PO. 04125 from the National Institute of Neurological

Disease and Blindness.

ii

 

 

 

 

 

'-' Pct-1
" ‘ V‘ ' i-

v

 

 

 

TABLE OF CONTENTS

LIST OF TABLES I O O O O O O O O O O O O I O O O 0
LIST OF FIGURES O 0 O O O O O O O O 0 O 0 O O O 0
INTRODUCTION 0 O O O O O O O O O O O I O O O O O 0
LITERATURE REVIm O O O O O O O O O O O O O O I I
General Metabolism .
Lens 0 O I O O O O O
Cornea . . . . .
Nutrition . . . .
Metabolism . .

“TERIALS AND METHODS O O O O O O O O O O O O O 0

Experimental Animals
Removal of Tissues .
Isot0pe Studies . .
Apparatus . . . .
General Procedure . . . .
Procedures for Studies wit Antimycin A
Procedure for Temperature Studies .
Procedure for Counting Radioactivity
Statistical Analysis . . . . . . . .
Microrespirometer Studies . . . . . .
Apparatus . . . . . . . . . . . . .
Measurement of Oxygen Consumption .
Comparison of Results with the
Microrespirometer . . . . . . . . . . .
Analysis of Media . . . . . . . . . . . . . .

RESULTS I O O O O O O O I I O O O O O O O O O O 0

General Aspects of Metabolism of Corneal and
Lenticular Tissues . . . . . . . . . . . . . .

Production of C1402 from 2-Cl4-pyruvate: Effect
Of Inhibitors O O I I O O O O I O O O I O O 0

Formation of Labeled CO from G-l-C14 and

2
c-e-cl4

iii

Page

vii

49

49

50

 

 

 

 

 

Untreated
Effect of
Effect of
Effect of
Effect of
Effect of

Effect of Inhibitors on Tissue C14
After Incubation with G-l-C

Tissues
Cyanide
Anoxia

Antimycin A In-vivo and In-vitro
Temperature

 

Capsule Damage

14

Effect of Inhibitors on the C14

Tissues After Incubation with 2-C

DISCUSSION

CONCLUSIONS

LITERATURE CITED . .

APPENDIX I

APPENDIX II

APPENDIX III

Incubation Solutions

Counting Radioactivity

Tables

iv

Activity

Activity in

-pyruvate

Page

50
51
52
53
55
59

59

61
63
76
78
84
86
89

 

. ..J . .-
. TO“. A

 

 

 

 

 

LIST OF TABLES

Table Page

1. Effect of inhibitors on production of
C1402 from 2-Cl4-pyruvate by ocular
tissues of trout at 13 C . . . . . . . . . 50

2 from G-l-Cl4 and

G-6-C by untreated tissues in PBS
at 13 C I I O I I '0 O O O I I O I O I I I 51

2. Comparison of C140

3. Effect of NaCN (10"3M) on c1402

from G—l—C and G-6-C by tissues in
PBS at 13 C I D I O O I I O I O I O I I I 52

production

4. Effect of anoxia on C140 production from

2
G—l-C14 and G‘6—Cl4 . o n o o o n c u o c 53
5. Effect of Antimycin A in-vivo and in-vitro
on C1402 yield from G-l-C14 and G—6-C14. . 54

6. Effect of temperature on C1402 production

from G-l-Cl and G-6-C from tissues

in PBS at 13 C . . . . . . . . . . . . . . 56
7. Effect of capsule damage on C1402 production

and Cl4 tissue content of lens incubated

in c—1-cl4 . . . . . . . . . . . . . . . . 59

8. The C14 activity in ocular tissues after
incubation with G—l—Cl4 and various
inhibitors . . . . . . . . . . . . . . . . 60
9. Effect of inhibitors on C14 activity in

ocular tissue after incubation with 2-C -
pyruvate I O I I I I I I I I I I I I I I I 61

 

 

"er—

 

 

 

 

 

Table Page

10. Comparison of C1402 production from
bilateral ocular tissues using uniformly

labeled glucose-C14 o o o o o o o o o o 0 89

11. Comparison of 002 values obtained from
constructed respirometer with literature
values for liver slices . . . . . . . . . 39

12. Metabolic data from respirometer studies. . 90

vi

 

 

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Figure

1.

LIST OF FIGURES

Changes in carbon atom distribution
during metabolism of glucose . . . . . .

Closed incubation system for collection

14
Of C 02 I I I I I I I I I I I I I I I I

Shaking apparatus containing incubation
vessels . . . . . . . . . . . . . . . .

Individual respirometer unit . . . . . . .

The effect of changing temperature on the

C1402 formation from G-l-C14 and G-6-C14

from the lens and cornea incubated
in PBS I I I I I I I I I I I I I I I I I

Sample channels ratio quench correction
curve for varying degree of quenching .

vii

 

Page

29

33
41

58

88

 

 

 

 

INTRODUCTION

Hatchery reared lake trout are known to develOp

corneal and lenticular lesions during and after the second

year of life which makes them useless for stocking lakes

(Allison, 1963; Hoffert and Fromm, 1965). A similar con—

K'l. '.- ‘...-l" .I". A

dition is known to exist in hatchery reared rainbow trout.

 

These lesions, which usually involve keratoconus, corneal
Opacity, and cataracts, closely resemble pathological
conditions of mammalian species.

The low oxygen demands of poikilothermic ocular
tissues make them well suited for in-vitro studies. In
addition, the animals are readily available, and offer a
large ocular tissue weight to body weight ratio which
makes tissues from even small fish acceptable for exper-
imental purposes.

The lens and cornea are avascular, therefore their
metabolic needs must be satisfied from the aqueous and
vitreous environment. Thus, information derived from
experiments using these tissues in-vitro in media similar
to their natural environment probably reflects the activity
which occurs under in-vivo conditions. Through similar
reasoning, one would also expect their metabolic activity

to be low.

J |

 

 

A comparison of the oxygen consumption and glucose

utilization of mammalian ocular tissues supports this con-
clusion. However, there must be a minimum amount of
energy needed to maintain viability. Certainly, the
primary function of the crystalline lens and cornea is to
retain their transparency. Although we do not have a
complete understanding, it appears that the maintenance

of lens and cornea transparencies depends on the utiliza-
tion of biological energy.

The free fatty acid content of the lens is
extremely low similar to that of the cornea (Krause,
1935). The aqueous, which serves as a nutrient media for
the two ocular tissues, is also low in free fatty acids.
Thus, unlike many other tissues, the lens and cornea are
unable to derive little, if any, energy from the oxida—
tion of free fatty acids, hence, the importance of glu-
cose as the major substrate for the production of energy
in the lens and cornea.

Carbohydrate metabolism in these tissues is
restricted by the nature of their environment. The
aerobic phase of glucose metabolism would not be expected
to play an important role in tissues which are avascular
and exist in an environment of low oxygen content. Even
if oxygen were readily available, these tissues must

remain colorless as well as transparent; therefore, they

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cannot contain large amounts of pigmented oxidative

enzymes of the cytochrome system or those of the ribo-

flavin containing group.

This study is an endeavor to provide a knowledge

of normal metabolic function which is the basis for

pathological investigation by answering the following

questions:

lI

2.

Is transparency a function of metabolic energy?
Is the tricarboxylic acid cycle (TCA) functioning
in the lens and cornea of rainbow trout?

Does the hexosemonOphosphate (HMP) shunt operate
in the lens and cornea as an alternate pathway
of energy production?

If present, what are the activities of the
respective pathways in the catabolism of glucose
for the production of energy?

What effect does temperature change have on the
activities of the various pathways?

What is the mechanism of entry for glucose and

pyruvate into the lens and cornea?

 

 

 

 

LITERATURE REVIEW

General Metabolism

 

Glucose is metabolized anaerobically in the
mammalian lens and cornea via two main pathways:
anaerobic glycolysis (Embden—Meyerhof pathway) and the
hexosemonOphosphate shunt (HMP). The Embden-Meyerhof
glycolytic pathway is familiar to most peOple and will
not be described in detail. Glucose-6-phosphate (G-6-P)
undergoes a series of steps to form pyruvic acid.
Pyruvic acid in the presence of an active citric acid
cycle (TCA) is completely oxidized to CO2 and water
(Figure l), but where the oxygen tension is low, or an
inactive TCA cycle exists, it is reduced almost exclu-
sively to lactic acid by means of an active lactic
dehydrogenase and DPNH formed at an earlier stage
(Langham, 1954). Fonner, Hoffert and Fromm (1969) have
found that ocular tissues of rainbow trout contain an
active lactic dehydrogenase.

As the name indicates,‘anaerobic glycolysis can
and does Operate in the absence of molecular oxygen. The
HMP shunt actually involves a dehydrogenation of G-6-P

and oxygen is not directly utilized in the reaction.

 

 

 

 

 

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Figure l.--Changes in carbon atom di
during metabolism of glucose.a

GGP
XuSP
R5P
S7P
GAP
E4P

HGP

Glucose-6—phosphate
Xylulose-S-phosphate
Ribose-S-phosphate
Sedoheptulose-7-phosphate
Glyceraldehyde-3-phosphate
Erythrose-4-phosphate

Hexose-G-phosphate

as. Hollman. 1964. Non-glycolytic
metabolism of glucose (Academic Press, New 1

 

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However, the eventual oxidation of the reduced TPNH
(which carries the hydrogen removed from G—6—P) may
require oxygen directly or indirectly.

The first step in the HMP shunt, the phosphory—
lation of glucose to G—6-P, is common also to anaerobic
glycolysis. This compound is then oxidized in two stages
with the concomitant reduction of the coenzyme triphos-
phopyridine nucleotide (TPN) occurring during both

reactions. The first reaction is a simple oxidation of

 

G—6-P to 6-phosphogluconic acid. Oxidative decarboxyla-
tion then takes place at carbon 1 of 6-phosphogluconic
acid to yield ribulose—S-phosphate and to liberate CO2
(Figure l). The ribulose-S-phosphate goes through a
series of intermediate steps eventually becoming D-glycer-
aldehyde-3-phosphate or fructose-G-phosphate. These
compounds are an integral part of the Embden-Meyerhof
pathway and offer a reentry to it from the HMP shunt.
The stbichiometric analysis of the shunt may be
illustrated in the following equation:

6G—6—P + lZTPN + 5G-6-P + Pi + 6C02 + lZTPNH + 12H+

Thus, the shunt yields CO and an abundance of TPNH as

2
its primary products.

While the reoxidation of DPNH is directly associ-
ated with the generation of ATP by the mitochondria,

there is as yet no known mechanism whereby TPNH can be

 

directly oxidized via a similar cytochrome system with

the concurrent production of ATP.

Lens

 

Interest of early investigators was centered on
the optical pr0perties of the lens and perhaps the first
important work on its integrity was that of Wagemann
(1891). After ligation of the posterior ciliary arteries

he observed permanent histological changes of the lens

 

and concluded that the integrity of the lens depended on
a continuous supply of oxygen, of nutrients, or both.
Since the lens is avascular, all its needs must
be supplied by the aqueous and the vitreous body. The
P02 of the aqueous in mammalian species is considerably
lower than that in the blood (Heald and Langham, 1956).
This limits the availability of oxygen to the mammalian
lens and other tissues supplied by the aqueous. But,
Fairbanks (1969) recently has found that the rainbow
trout eye concentrates O2 to a level of 445 mm Hg P02
measured behind the retina. A gradient was present and
showed a P02_of 103 mm Hg in the vitreous body.
Sippel (1962) confirmed Kleifeld and Hockwin's
(1959) statement that oxygen uptake by the lens is
02 in the media, and extended it
to say that lens respiration increases abruptly to plateau

directly dependent on P

at a level of 140-230 mm Hg P0 in dry gas, then rises
2

 

continuously through to 730 mm Hg P He suggests that

0 .
this biphasic curve may indicate thai two oxidation
reactions may be involved. Kronfeld and Bothman (1928),
Fisher (1931), Michail and Vancea (1932) all observed
that anaerobic as well as aerobic glycolysis occurs in
the mammalian lens to supply the needed energy.

The use of various known metabolic inhibitors

expanded the information on oxygen consumption of the

lens.

 

Data of Field gt_al (1937) suggest that 2,4,-
dinitrophenol may be the etiologic agent in the develop-
ment of cataracts in a small percentage of cases after
its therapeutic administration. Ely and Robbie (1950)
exposed the lens to various levels of cyanide in the
incubation media. A concentration of 10-4M HCN inhibited
oxygen uptake by 50%, but about 9% of the respiration of
the rabbit lens is resistant to a level of 10_2M HCN.
They believe that one mole of cyanide inhibits one mole
of the active heavy metal containing enzyme of the
crystalline lens.

Attention was shifted by the investigators from
the measurements of gross metabolic activity to experi-
ments designed to elucidate and assay intermediary
metabolites of different metabolic pathways used to

produce energy. Glucose, the major substrate for the

 

 

 

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10

production of energy, must first pass through the lens
capsule and then encounter the second biological barrier
consisting of the cell membrane. Studies by Harris,
Hanschild and Nordquist (1955) on accumulation of glucose
within, and utilization of glucose by rabbit lenses
treated with various enzyme inhibitors indicate that
glucose does not enter the lens solely by simple diffu-
sion but also by some active process. They think that one
site (or barrier) at which this process occurs lies at or
in close proximity to the lens capsule and there may be
more than one barrier. Experimenters have also attempted,
without success, to establish the role of insulin in the
transport of glucose in the lens. Levari, Wertheimer
and Kornbluth (1964) investigated the effects of various
concentrations of glucose in the media on metabolism.
Incubation of intact rat lenses in increasingly higher
concentrations of glucose resulted in a proportional
increase in liberation of C1402. Lactic acid production
was stimulated as glucose concentration increased up to
1.0 mg/ml but higher concentrations had little affect.
Once inside the cell, glucose is phosphorylated
by the hexokinase reaction which involves the utilization
of ATP. The product, G-6—P, can then directly enter the

HMP shunt with the resultant removal of the first carbon

atom and the formation of a five carbon residue, or it

 

 

 

 

11

can undergo transformation to its isomer, fructose-6-
phosphate, and continue in the glycolytic pathway.

In 1955 Green, Bocher and Leopold published the
first of several articles describing the fate of glucose
during anaerobic metabolism of the crystalline lens.
They concluded from their experiments that the necessary
intermediates for the formation of lactic acid from
glucose were present in the lens, and that one of the
rate limiting steps for the pathway was the limited amount
of hexokinase enzyme present in the tissue.

Recognizing the fact that the lens has a low
oxygen consumption does not allow one to make any a
priori assumptions as to the utilization of glucose by
the lens. According to Ely (1951), lactic acid, pyruvic
acid, and various intermediates of the citric acid cycle
were oxidized when used as substrates for bovine lens
homogenates. However, the addition of cytochrome C was
essential for maximal aerobic oxidation. This is the
first evidence pointing to the low activity of the citric
acid cycle in the lens. Harris, Hanschild and Nordquist
(1954) proved that oxidation is essential for the main-
enance of viability of the lens. This information led
Kinoshita (1955) to investigate the effectiveness of the
citric acid cycle in aerobic metabolism. He speculated
that if the function of the TCA cycle is to completely

oxidize pyruvate to C02, an indication of the activity of

 

 

 

 

 

 

 

12

this cycle might be gained by studying how effectively

the lens would oxidize 2-C14-pyruvate to C1402. Using

14 . . . .
2-C -pyruvate, the radioactive carbon is incorporated
in the middle of this three carbon compound and thus the
yield of C1402 is taken as an index of the complete

oxidation of the pyruvate. He found that bovine lenses

.x—HH'nl

were unable to utilize 2-C14-pyruvate to any appreciable

extent during four hours of incubation and concluded that

 

the citric acid cycle is relatively inactive in the lens.
In the second part of the experiment Kinoshita
applied the fact that the metabolic fate of the carbon-l

and carbon-6 atoms of glucose differs depending on how

 

 

 

 

 

glucose is metabolized (Figure 1). If glucose were

metabolized via the glycolytic and citric acid pathways
exclusively, the carbon atoms 1 and 6 of glucose become
the methyl carbon of pyruvate and subsequent oxidation

by the citric acid cycle would result in C-1 and C-6 of

glucose appearing as CO2 simultaneously. If glucose were

metabolized via the HMP shunt, the C-1 atom of glucose

would appear as CO2 much earlier than C-6, since there is

preferential cleavage of the C-1 of glucose. To test this

approach, the lens was incubated with either glucose-l-

C14 14 (G-6-C14) and the amount

(G-l-Cl4) or glucose—G-C
of C1402 produced was compared. Results from three

different mammalian species, cat, rabbit, and bOVine

14 14
established that a G-l—Cl4/GeéaC ratio of C 02 recovered

 

13

was approximated 40:1. Thus, the lens exhibits a
preferential oxidation of the C-1 of glucose to C02,
which strongly suggests the participation of the HMP
shunt.

A more quantitative estimate may be made by
measuring the radioactivity incorporated in lactic acid
when labeled C-l or C-6 glucose is used as the substrate.
By the HMP shunt, the carbon-1 atom of glucose is directly
converted to C02. Therefore, it seems reasonable to
assume that the activity recovered in the lactate from
G-l-C14 is derived solely from glycolysis. Conversely,
the radioactivity recovered in lactate from G-6-C14 is
that contributed from both the direct oxidative and
glycolytic pathways. The ratio of C14-lactate from

14 is the approximate fraction of glucose

G-i-c14/c-6-c
metabolized via glycolysis. Kinoshita's (1955) value
for the bovine lens was 0.79. This means that for every
four molecules of glucose utilized by glycolysis, one
molecule of glucose is metabolized via the HMP shunt.
Kinoshita and Wachtl repeated the eXperiment in

1958. This time one lens per flask was incubated for

The results were similar. Labeled

. 14
was formed at a rate 44 times faster uSing G-l-C

twenty-four hours.

CO2

than when G—6-Cl4 was used. The radioactive lactic acid

data demonstrates that 14 percent of the glucose was

oxidized by the direct oxidative pathway.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

am
Calls
Indic

“0311a;

 

14

The use of differentially labeled radioactive
glucose, as mentioned above, provided a needed tool in
the research of carbohydrate metabolism. Lerman's (1961)
interest was in the formation of metabolic pathways
during development and the relation of the age of the
animal to carbohydrate metabolism. The C-l/C-6 ratio of
C1402 from the lens in his experiments showed marked
change correlated with age as did the TPNH/TPN ratios.
The DPNH/DPN ratio was similar in both young and mature
rat lens. The conclusion was that_the HMP shunt dimin-
ished considerably in activity in the lens as the animal
ages. Later, Lerman, Donk and Pitel (1962) established
with fetal rat lenses that aerobic glycolysis and HMP
shunt of glucose oxidation are both functioning on the
17th day of gestation, but the TCA cycle is not operative
until 20-21 days. A steady increase in amount of lactic
acid per lens from day 17-21 indicates that glycolysis is
functioning.

What happens to the metabolic scheme of carbohy-
drates in a pathological tissue? Lerman (1959)
experimentally induced cataracts by two different methods.
He fed rats a high proportion of galactose in the diet,
and in 1962 he produced cataracts following an alloxan
caused diabetic condition. The radioactive CO2 data
indicates that the oxidation of G—6-Cl4 occurs at about

normal rate or may increase slightly. Carbon dioxide

 

 

15

from G-l-C14 is less than normal after four days of either
treatment and remains low.
In 1965 Van Heyningen reported the separation of
ten substantially labeled compounds of various metabolic
pathways from protein free extracts of incubated lenses.
Her technique involved the use of electr0phoresis, paper
chromatography, and radioautography. In the absence of

oxygen the radioactivity was prevalent in lactic acid, .

 

alpha-glyceroPhosphate, and glycerol. Aerobic conditions

‘5‘

caused the radioactivity of the lens proteins to be 2-4
times greater than in the absence of oxygen. Finally,
the radioactivity of all labeled compounds was predom—
inant in the outer part (cortex).

In the mammalian lens the biological energy
necessary for the maintenance of transparency and repair
is supplied primarily by the reactions that break down
glucose to lactic acid. To enter the lens from the
aqueous humor, glucose must first cross a barrier,
believed to be located in the area of the capsule or
epithelium, at the expense of energy. Once inside the
low levels of enzymes associated with the TCA which
aerobically oxidizes glucose restricts the lens metabolism

mainly to anaerobic glycolysis, the low P in aqueous may

0
2
also restrict the TCA cycle. The HMP shunt is functional

and although it produces most of the CO liberated by the

2
mammalian lens, only a minor part of the total glucose

 

blc

exi:
is g
tens

(Fai

Nutr

remc

cart

the

the 1

way 0
kinet
the 1
blood

COde,

 

16

used is metabolized by this pathway. Lactic acid which
is produced in the lens freely diffuses into the aqueous
where it is present in higher concentrations than the
blood.

The trout lens differs from mammalian lenses by
existing in an environment with an oxygen tension which
is greater than that of the blood instead of low oxygen
tension (compared to blood) found in mammalian eyes

(Fairbanks, 1968).

 

Cornea

Nutrition

The basic metabolites of the cornea, glucose and
oxygen are of great interest. Equally important is the
removal of breakdown products of metabolism, particularly
carbon dioxide and lactic acid. These substances can
move into and out of the cornea across three surfaces:
the anterior and posterior surfaces of the cornea, and
the corneal scleral junction called the limbus.

How much cf the needed materials are supplied by
way of the limbus is a moot point. According to the
kinetics for diffusion of sodium, at a point 6 mm from
the limbus, 99% of that entering the cornea from the
blood will be lost to the aqueous humor. The kinetic

conditions for the movement of glucose and oxygen are

 

 

 

 

51)

1a}

meet

this

 

 

17

less favorable than for sodium. These calculations are
made without regard to the amount that will be metabolized
along the way. It is unlikely that any of the nourishment
from the limbal blood vessels ever reaches the center of
the cornea.

The rear surface of the mammalian cornea is

 

bathed by the continuously circulating aqueous. The
anterior surface is covered by a thin film of tears which

separates it from the air. The environment of the

 

anterior surface of the teleost cornea is the water in
which it lives. Whereas the mammalian cornea may be
supplied to some extent by its tears, the environment is
unlikely to contribute much to the metabolic needs of the
teleost cornea. In fact, the hypotonic environment of
freshwater species places a great osmotic burden on the
cornea.

Measurements on the excised rabbit cornea hint
that the primary resistance to the diffusion of oxygen
is within the stroma (Heald and Langham, 1956). From
this it has been calculated that the oxygen diffusing
into the cornea from the aqueous humor is inadequate to
supply the respiratory requirements of the epithelial
layer., This leaves only the anterior supply route to
meet the oxygen needs of the epithelium. For mammals,

this seems reasonable partly because of the large pressure

 

18

gradient of oxygen between the surrounding air and the
cornea, and partly because of the ease of diffusion of
oxygen through the tear layer.

Noting that water has no glucose, and regarding
the limbal supply as insignificant, the sole source of
glucose to the teleost cornea is the aqueous humor. The
concentration of glucose in the rabbit aqueous is
approximately 100 mg% (Giardini and Roberts, 1950). This

is not greatly different from that in the tissue fluid of

 

the stroma. Consequently, there must be some mechanism
other than simple diffusion across the endothelium to
satisfy the cornea. The small amount of glucose contri-
buted by tears is inadequate, so the aqueous appears
responsible as the major supply route to the epithelium
of the mammalian cornea.

Carbon dioxide and lactic acid are the main
metabolic end products in the cornea and both appear to
be eliminated by simple passive diffusion. Carbon
dioxide, like oxygen, is fat soluble in its unionized
form and consequently it penetrates cellular membranes
readily. Because most of the carbon dioxide is formed in
the epithelium, it is likely that the anterior surface of
the cornea would be the primary escape route (Redslob and
Trembley, 1933).

A lactic acid concentration gradient (of about

25 mg%) exists favoring the stroma of the cornea over the

 

 

 

19

aqueous humor. According to Langham (1954), this
gradient is sufficiently large to permit the unmetabolized
lactic acid to leave the cornea by passive diffusion

across the endothelium.

Metabolism

Respiration of the cornea, like that of the lens,
has been measured numerous times under various conditions.

The results agree well. The oxygen uptake by the cornea

 

does not seem to vary when subjected to different condi-
tions. Langham's (1954) results agreed even when incuba-
tion proceeded in the absence of a liquid incubation
medium. Figures for the respiratory quotient seem to
point toward carbohydrate utilization exclusively, until
challenged recently by Haberich and Dennhardt (1965).
They reported an R.Q. of 0.8 which would indicate sOme
utilization of protein and possibly some fat as a source
of energy.

Langham (1952) denuded the cornea and measured
the respiration of its parts. Respiration for the
epithelium and perhaps the endothelium is comparable to
that of some of the more active tissues of the body.

The stroma in the rabbit and in the ox can be considered
relatively inert. Pontocaine, chlorbutanol, cyanide,
iodoacetate, pentobarbital, atropine, and cocaine in

varying degrees of effectiveness all inhibit the oxygen

 

 

it:

sub

 

20

uptake of the bovine cornea (Herrmann, Moses, Friedenwald,
1942). Kinoshita (1959) later established that ponto-
caine blocked the citric acid cycle and HMP shunt, but
did not affect anaerobic glycolysis.

Two pathways are established for metabolism of
glucose in the cornea: anaerobic glycolysis supported
to some extent by the TCA cycle, and the HMP shunt.

The aerobic pathway of glucose metabolism in the
cornea has been investigated by Kinoshita and Masurat
(1959). From the relative rates of oxidation of the
three carbon atoms of pyruvic acid, it appears that the
TCA cycle is functioning in the cornea. However, its
role is limited, evidenced by the large accumulation of
lactate in-vivo and by the relatively small amount of
pyruvate oxidized by this tissue. Lactic acid that is
not oxidized diffuses out of the tissue.

Anaerobic production of lactic acid in rabbit and
bovine corneas with a sufficient amount of other necessary
nutrients available is about four times greater than the
aerobic production (Herrmann and Hickman, 1948c; Langham,
1954). The major portion occurs in the cellular
epithelium and the endothelium. The stroma has a low
rate of production. The denuded cornea retains about one-
third of its ability to oxidize lactate but loses 90% of
its capacity to oxidize glucose when incubated with these

substrates in radioactive form (Kuhlman and Resnik, 1959).

”hummus t.t't3.n.'¢‘.\.t$»‘t.§uk‘o'r$‘&b.aJ. .. a, .

  

nWJ'I'fv 074

AP 1‘ 5:“; I.‘.l~ 9‘31?) '4 1"“...‘91'

- .~ It
«V,

  
   
    

V7.37]: n .2 (1"'u'a-'-_~'

5‘34""...5'5" "NJ"

' " .lf"I-n-r ~'-_~

 

[)0

the

gill

 

21

In 1964 Kinoshita and Masurat produced evidence
for the HMP shunt in bovine corneal epithelium. Measure-
ments of the activity of glucose-6-phosphate and 6-
phosphogluconate dehydrogenases, the formation of pentose
phosphate and carbon dioxide from phosphogluconate, and

the heptose formation from pentose phosphate convlusively

 

established the pathway. Kinoshita, Masurat and Helfant
(1955) quantitatively estimated the contribution of the
HMP shunt to the total oxidation of glucose. They
reported that 65% of glucose was metabolized via the
conventional glycolytic scheme, and 35% by the shunt.
Four years later, Kuhlman and Resnik (1959) indicated
that even more, up to 70% of the oxidation of glucose by
the cornea proceeds by the HMP shunt. They also added a
large amount of unlabeled lactate, anticipating that the
specific activity of the intermediates beyond this step
would be reduced. The time interval was brief enough to
prohibit the system from attaining an equilibrium. The

radiochemical yield of C140 from G—6—Cl4 decreased 86%

2
and that from G-l-Cl4 only fell 30%. This indicates that
the predominate pathway of CO2 production from carbon one
of glucose does not pass in equilibrium with the lactate
pool.
How are the various pathways related and what is

the mechanism of control designating the method of

glucose oxidation to be used? The participation of the

 

 

 

22

direct oxidative pathway in the utilization of pyruvate

was demonstrated by studying the effect of it on the

anaerobic oxidation of G-l-Cl4 by corneal epithelium

(Kinoshita, 1957). Addition of unlabeled pyruvate to the
14

incubation media stimulated the oxidation of G-l-C to

an eight fold increase in the production of C140 over

2
that produced from media lacking additional pyruvate.
Increase in the 2-Cl4—glucose oxidation to C1402, but not
that of G-6—C14 added additional support for the involve-
ment of the HMP shunt in the utilization of pyruvate.
There seems to be an interrelation between the conversion
of pyruvate to lactate and the TPN-linked dehydrogenases
of the direct oxidative pathway seems like the probable
explanation for the stimulation of the C-1 oxidation of
glucose. Lactic acid dehydrogenase of the corneal
epithelium seems capable of functioning to some extent
with TPN; however, DPN is the preferred coenzyme
(Kinoshita, 1957).

The results indicate that C-l oxidation of
glucose to carbon dioxide under anaerobic conditions in
the presence of pyruvate is greater than that under
aerobic conditions without added pyruvate. It therefore
appears that in the corneal epithelium, the rate of re-
oxidation of TPNH is the rate limiting step in the HMP

shunt.

 

23

Maintenance of constant water content in lens and
cornea is an energy consuming process dependent on meta-
bolic activity. Since a shift in water content of the
tissue is an important process in corneal pathology, a
decrease in metabolic production of energy is indicated
(Schwartz, Danes and Leinfelder, 1954). -The type of
process for maintenance of hydration or the site of
action is unclear; however, it may be associated in some
way with the maintenance of transparency. Smelser (1961)
thinks that control of corneal hydration in fish is
different from mammals. The elasmobranch cornea is not
hydr0philic and does not swell in any aqueous media.

The degree of swelling is greater in fish adapted to the
sea and less in carp which live in a hypotonic medium.
Since these poikilothermic tissues do not show a tempera-
ture reversal effect, perhaps there is no metabolic
control. He offers the explanation that the swelling
pressure is balanced by a high colloid osmotic pressure
consisting largely of mucopolysaccharides.

Antimycin A was introduced by Strong (1958) as a
potential systemic antifungal agent. However, when it
was found to inhibit cytochrome C of the electron trans-
port chain its systematic use was limited, and it is now
marketed as Fintrol (Ayerst, New York) for use by fish

conservationists as a fish eradication agent in lakes

 

 

 

 

 

 

24

before restocking. Rodonski and Wendt (1966) and Foye
(1969) noted that one of the effects of the compound on
many of the dying fish was the presence of cloudy eyes.

In summary, the cornea also appears to utilize
glucose exclusively which enters primarily from the
aqueous through the posterior surface of the tissue.
Carbon dioxide and lactic acid, both metabolic end pro-
ducts, appear to be eliminated by simple passive diffu-
sion. Once within the cells, glucose is phosphorylated
by the hexokinase enzyme then can enter either the
glycolytic or HMP shunt pathways, which are both functional
in mammalian corneas. The TCA cycle has a limited ability
to oxidize the products of glycolysis, lactate and pyru-
vate, to C02. The lactate that is not oxidized diffuses
out of the tissue. Anaerobic production of lactic acid
in mammalian corneas is about four times greater than the
aerobic production of lactate.

The HMP shunt is active and may account for up to
70% of the metabolism of glucose by the mammalian cornea.
The rate limiting step of the HMP shunt seems to be the
rate of reoxidation of TPNH which is a primary product of
the pathway. Lactic dehydrogenase of corneal epithelium
seems capable of functioning to some extent with TPNH;

however, DPNH is the preferred coenzyme.

 

 

 

 

MATERIALS AND METHODS

EXperimental Animals

 

The rainbow trout (Salmo gairdneri) used in these

 

experiments were obtained from the Michigan Department of
Natural Resources at Grayling, Michigan. Trout approx-
imately two years old, weighing between 100—250 g, were
selected. The fish were transported from the hatchery
to the East Lansing campus in a galvanized metal tank
lined with non-toxic paint. The tank was housed in a
polystyrene-lined plywood box to maintain constant water
temperature, and was fitted with an agitator for aeration.
The laboratory holding facilities consisted of
fiberglass-lined plywood tanks supplied with a continuous
flow of dechlorinated water at one end with an overflow
spout at the other end. The photOperiod was 15 hours
light and 9 hours darkness each day and the water
temperature was 13 C. Constant aeration was provided by

activated charcoal-filtered air lines.

Removal of Tissues

 

Fish were killed by a sharp blow to the top of

the head. With the fish on its side, a small incision

25

 

.1

' ‘ 4 '. .l .o-o. "0"“?‘3 s

 

 

st]

we:

the

eXpe

lens

 

26

was made in the ventral limbal area of the cornea with
one blade of an iris scissors. Rat tooth forceps were
used to hold the cornea fixed at the initial incision,
while the cornea was cut around the entire periphery at
the limbus anterior to the iris. The excised cornea was
placed in iced cold blooded Ringers solution (Appendix 1). EB”
Small curved forceps were then placed under the lens

which was gently lifted out of the eye and also placed in

 

the iced Ringer bath. lj
The tissues were carefully washed in the iced
solution, blotted dry on No. l Whatman filter paper,
weighed on a Roller-Smith precision balance to the nearest
0.1 mg, and placed in the apprOpriate media. Suspensory
ligaments attached to lenticular tissue were removed with
a scalpel during the blotting process. Damage to the
lens capsule was infrequent but visibly manifest. If
damage occurred, all tissues from that fish were discarded.
The entire blotting and weighing procedure deprived the
tissues of media for less than one minute. Surgical in-
struments, syringes, needles, center wells, and media
were all sterile.
Weights of the tissues ranged from 80-120 mg for
the lenses and 40-60 mg for the corneas, but in any one
exPeriment, the variation in weight between contralateral

lenses or between corneas was 2.0 and 4.0 mg respectively.

 

 

27

Isot0pe Studies

 

Apparatus

 

Liquid scintillation counting vials of 20 ml

capacity were used as the closed incubation system. The

apparatus was modified from the design of Hostetler gt_al F

(1966) to include a No. 2 black rubber stopper inserted

in the neck of the vial to complete the enclosure. The

rubber stOpper also served as support for the center well. .
L

 

A local glassblower constructed the center well from
pyrex glass to have a volume of approximately 2.0 ml. An
18-gauge needle 2 inches long and fitted with a 3-D (No.
M809) spring loaded stOpcock (Beckton, Dickinson and Co.,
Rutherford, N.J.) was permanently inserted through the
stopper to provide an entrance to the center well for the
addition of acid (Figure 2).

The tissue and media were contained within the
center well suspended from the stOpper. This allowed the

CO absorber to remain in the bottom of the counting vial

2

after removal of the center well. The possibility of
error encountered during transfer of the CO2 absorber in

other procedures was eliminated.

General Procedure

 

In all exPeriments chemically defined Phosphate-

Buffered-Saline (PBS) obtained from the Grand Island

 

 

'—r

 

 

 

 

28

Figure 2.--Closed incubation system for collec-
tion of C O .

2
1. Liquid scintillation vial (20 ml).
2. Glass center well for tissue and media.
3. Black rubber stopper (No. 2).
4. Syringe needle (18 gauge, 2.5" long).

5. B-D spring loaded stopcock (No. M509).

 

29

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

\

 

 

 

Figure 2

A

 

 

 

 

 

 

 

30

Biological Co., Grand Island, New York, was used as
incubation media for the tissues (Appendix 1). A 0.4 ml
volume of 5 gm/100 ml dextrose (Abbott Laboratories,
Chicago, Ill.) was added to 19.6 ml of PBS resulting in
a concentration of 100 mg/100 ml media. The final con-
stituent of the media was Suc of a Cl4 labeled compound.
Each center well contained 0.7 m1 of incubation media,
and all solutions used were sterile.

After the tissues were added to the center wells,

 

the entire chamber was flushed with 100% oxygen for
approximately 10 seconds and then the vessels were sealed
and shaken for 4 hours at the desired temperature.
Shaking was accomplished by a cam-driven 24 cm diameter
circular table which oscillated about its center. The
cam was cut with an instantaneous decceleration phase
which caused the spring loaded table to rapidly return
and strike a rubber stopper, thereby creating agitation
of the media. The distance the table was allowed to
return was the maximum allowable without Splashing media
from the center well and was empirically determined to
be approximately 1 cm at the periphery of the table. The
cam speed caused about 50 impulses per minute, and the
table held 12 incubation chambers.

At the termination of the incubation period, 0.2
ml of Hydroxide of Hyamine lO-X (Packard Instrument Co.,

Downers Grove, Ill.) was injected into the liquid

 

 

31

scintillation vial through the side of the stOpper

14

thereby preventing loss of C 0 Initially, 1.0 m1 of

2I
l4

Hyamine was used to absorb the C O in the vial, but

2
successively reducing the amounts to 0.2 ml showed that
this amount was adequate to absorb all CO2 produced. This
provided a saving in materials with much less quenching of
the counting solution.

The reactions were terminated at this time by
injection of 0.1 ml of 0.67 N HCl through the 18-gauge
needle into the center well. To correct for the approx-
imately 0.1 ml dead space in the needle, acid was
injected into the needle by a 1.0 ml tuberculin syringe
until it was visible at the needle tip. Then an addi—
tional measured 0.1 ml was injected from the syringe into
the center well. The vessels were shaken for an additional
hour to absorb into the Hyamine the C1402 evolved. The
center wells containing the tissue and incubation media
were then removed and treated as described below.

Fifteen milliliters of a liquid scintillation
counting solution were added to each vial and the C14
activity was determined with a Mark I Liquid Scintillation
System (Nuclear-Chicago Corporation, Des Plains, 111.).
Originally, counting solution I (Appendix 2) was used,
but later the ingredients were decreased to conserve

materials. Counting solution II (Appendix 2) was used in

the final eXperimentation and did not alter either

 

 

   

 

 

 

v— " J- 1'99"

32

Figure 3.--Shaking apparatus containing
incubation vessels.

.ning

 

33

 

 

Figure 3.

 

34

counting efficiency or water solubility for the procedure.
All scintillation chemicals and counting vials are pro—
ducts of Packard Instrument Co., Inc., Downers Grove,
Ill. The dioxane used was certified grade from Fisher
Scientific Co., Fair Lawn, New Jersey.

Corrections for the "carbonate like" material
(Merlevede, Weaver, and Landau, 1963) contaminating the

14

G-l-C were made by measuring the C140 released follow-

2
ing acidification of the media in control vessels that
had been incubated without tissues.

14

After collection of C O the tissues from each

2,
center well were removed, rinsed thoroughly in cold-
blooded-Ringer solution to elminate excess media, blotted
dry on filter paper, and placed in a scintillation vial
containing 1.0 ml of Hyamine Hydroxide. Solubulization
of the tissues was aided by heating to 60 C for 18 hours.
Finally, 15 m1 of scintillation cocktail were added to

each vial and tissue activity was counted.

A 0.1 m1 aliquot of media from two control center

wells without tissue and two randomly selected experimental

vessels was counted for activity to determine specific
activity (dpm/mg) of the media. All C14 compounds were
purchased from New England Nuclear Corporation, Boston,

Mass.

 

 

 

 

35

Procedure for Studies with
Antimycin A

 

 

Antimycin A was donated for experimental use by
the Veterinary Medical Division of Ayerst Laboratories
Incorporated, New York, N.Y. The compound is only slightly
soluble in polar solvents; therefore, it was delivered in F;
an acetone solvent. The acetone solvent from the orig-
inal solution was evaporated from a measured volume and

was replaced with 100% ethanol to make a concentration of

 

Ir"

2.6 ug/ul ethanol. One ml of the solution was added to
1.6 ml of sterile distilled water making a concentration
of l ug/ul 62% ethanol. This is the concentration of
water at which the inhibitor began to precipitate. Five
ul (5 ug) of this solution were injected in-vivo into the
anterior chamber of the left eye with a micropipet
attached to a microburet.

A control solution of 1.0 ml of 100% ethanol in
1.6 m1 of water without inhibitor was made and 5.0 ul
were injected into the Opposite eye. The eyes were
examined periodically for 72 hours after the time of
injection.

In-vitro paired exPeriments consisted of adding
Antimycin A to the media for the tissues of one side at
a concentration of 2.6 ug/ml. Both l-Cl4 and 6-C14

radioisotoPes of glucose were used in-vivo as well as

in-vitro.

36

Procedure for Temperature Studies

To determine the effect of changing temperature

on the tissue production of C140 from G-l-Cl4 and G-6-

2
C14, 4 hour experiments were conducted at temperatures
of 4, 13, 23, 33, and 43 C. The procedure for tissue
preparation was the same as described above, and to obtain
the desired temperature the shaking apparatus containing
the incubation vessels was placed in a Model 82 Fisher
Low Temperature Incubator (Instrument Division, Fisher
Scientific, Pittsburg, Pa.). The temperature of the
chamber was monitored using a YSI (Yellow Springs Instru-
ment Co., Yellow Springs, Ohio) Model 432 Thermistor and

Model 43TD Telethermometer. The temperature fluctuated

no more than 1 C of the desired temperature.

Procedure for Counting Radioactivipy

The Mark I Liquid Scintillation System was used

1 . . .
to count all experimental samples for C radioactiVity.

The amplifier gain (or attenuation) on channel C was ad-

justed to provide highest efficiency (counting rate) for
a given sample between the selected lower and upper dis-

criminator levels. The least quenched standard of a

series of quenched standards (Nuclear-Chicago Corporation)

was used for adjustment. The maximum machine efficiency

was 91% (see Operating manual for Mark I system for

details).

 

 

 

 

37

Counting efficiencies for all samples ranged
between 40-85% with the majority counted at 50-80% level.
Efficiency for the tissue activity was at the lower end
of the range due to the quenching effect of Hyamine which
turned yellow when heated to 60 C for 18 hours. The
C1402 counting efficiency was high because of the small
amount of Hyamine used. All samples were counted for 40
minutes or 100,000 counts, whichever occurred first. A
statistical error of less than 3% resulted from the
counting procedure (Operating manual for Mark I System).

To correct for varying amounts of sample quench-
ing, the upper level discriminator on channel B was
lowered so channel B monitored the lower 30% of the
counting rate of channel C. A channels ratio quench
correction standard curve was then plotted using the
series of quenched standards. Machine counting effic-
iency was plotted on the ordinate against channels ratio
(C14 cpm channel B/Cl4 cpm channel C) which are plotted
on the abscissa. With the channels ratio (B/C) printed
by the counter, the counting efficiency was obtained from
the quench correction standard curve (Appendix 2). All

counts were corrected to 100% efficiency (dpm).

_ Machine counts (cpm)
% efficiency

 

Total counts (dpm) x 100

 

 

 

38

Statistical Analysis

 

Statistical analysis of data from the paired
experiments used throughout was calculated using the non-
parametric Walsh test for small (N) sample numbers. The
term significant when used hereafter indicates a calcu-

lated p value of less than 0.05.

Microrespirometer Studies

 

Apparatus

 

A multiple-unit constant-pressure microrespir-
ometer was constructed for oxygen consumption studies.
Winterstein (1912) introduced the constant-pressure
principle and Scholander (1941) added modifications to
measure changes in gas volume with a micrometer—controlled
burette attached to each respirometer unit. Reineke (1961)
connected a series of respirometer units into a manifold,
permitting measurement of gas volume changes of all units
with a single micrometer—controlled burette to further
improve the design. Construction of the microrespirometer
was patterned after Reineke's design, and included the
following alterations:

1. A three-way stopcock was inserted to provide
a short circuit across the manometer, thereby eliminating
the possibility of ejecting the fluid from the manometer

when instantaneously subjected to a large pressure

_. egg"), . 4,
Ber-":9? £3119"?
‘ "v on: «1’97; y- =4

. .9
"'9 “'3‘ ,AA

 

In

co
co
ox;
for
Vide
Posi

flas

 

39

difference between the manifold and the thermobarometer
flask.

2. Nine respirometer units were included, per-
mitting the use of paired experiments with a blank unit
remaining.

3. Interchangeable syringe microburets were used
to assure maximum sensitivity.

4. Respirometer units, individually constructed
and connected to each other by ground glass ball-joints,
provided easy removal and exchange if damaged.

Each individual respirometer unit is basically a
U-tube manometer with one arm connected to an incubation
flask, and the other arm attached to a thermobarometer
compensating flask. A piece of glass tubing served as a
segment of a common manifold, and a connection was made
from this segment to the side of the manometer with the
incubation flask. A two-way stopcock was inserted in
this connection to separate the unit from the common
manifold, creating a closed incubation system. This
connection provided a route for addition of gas from the
common manifold to the incubation flask, replacing the
oxygen consumed by the tissue during incubation. The
forementioned three-way stopcock was introduced to pro-
vfixie a direct connection, when turned to the appropriate
'position, from the thermobarometer to the incubation

flask, by-passing the manometer. The unit was made of

 

 

 

‘, "7' ‘lwr'VEWW‘V. v

40

Figure 4.--Individual respirometer unit.

Manometer.

Standard 7/20 joint to connect incubatiori
flask.

Standard 14/20 joint to connect compensatitni
flask.

Three-way stOpcock.
Manometer by-pass.

Two-way stOpcock.

Segment of common manifold.

Standard 12/2 ball and socket joint.

 

 

 

41

 

 

ion

 

 

 

 

p

isatiO

 

 

 

Figure 4.

' 0
_ /

      

42

2 mm (i.d.) capillary tubing and stOpcocks having a 2 mm
bore. Standard taper joints of size 17/20 were used at
the incubation flask connection to accommodate Warburg
reaction vessels. A 15 ml Erlenmeyer flask used as the
compensating flask was joined to the unit by a 14/20 joint.
Manometer fluid (Kerosene, colored with Sudan III) was
injected into each manometer with a small-bore poly-
ethylene tube connected to a syringe. Manometers were

filled to 1 cm below the expansion bulbs, and zero points

 

were marked with narrow pieces of adherent tape on the
left arm of each manometer, level with the meniscus.

A local glassblower geometrically fabricated the
glassware to fit like an inverted U over an acrylic
plastic bench-type support. The 9 respirometer units
were coupled together by 12/2 ball and socket joints on
each manifold segment to form a common manifold. The
first unit on the left was fitted with a two-way stopcock
and the extreme unit on the right was fitted with a male
syringe adapter holding a 20-gauge needle. With all nine
units assembled in a row on the bench, flasks were
located at the rear for accessibility, and manometers
*were in front to facilitate visualization. Individual
rumits were firmly held to the acrylic plastic bench by

tension exerted from two pieces of rubber tubing stretched

over the two arms extending from the manometer and fastened

 

 

 

 

43

on both sides. The bench was mounted on wheels to allow
shaking motion.

During Operation, the bench with glassware in-
cluded, was completely submerged in a constant temperature
water bath, which was also constructed of acrylic plastic.
This tank rested on a plywood superstructure built with a
platform at each end of the tank. One platform rigidly
supported an electric motor (Bodine Electric Co., Chicago,

Ill.) used for shaking the bench. It was rated a 1/18 hp

 

"-—

at 5000 rpm, on line voltage, but a Powerstat (Superior
Electric Co., Bristol, Conn.) was inserted in the line

to regulate motor speed. The motor was connected to the
movable bench through a removable bronze rod, which was
fixed to an eccentric bronze ball-joint on the reduction
shaft of the motor, and extended to a similar metal joint
on a vertical plastic projection rising from the bench.
The joints were easily separated for removal of the bench
from the water. Mounted on the other platform was a
dial-type syringe microburet (model No. SBZ, Micro-Metric
Instrument Co., Cleveland, Ohio). A microburet syringe,
calibrated at 0.500 ul per micrometer division, yielded
Inaximum accuracy, but other syringes were interchangeable,
thus increasing versatility of the apparatus. Communica-
tixln from the microburet syringe to the common manifold

was through a small-bore polyethylene (PE100) tube, fitted

 

44

tightly over 20-gauge hypodermic needles on both the
common manifold and the microburet syringe.

Water bath temperature was maintained by keeping
the entire apparatus in a controlled temperature room at
13 0.5 C. Space was provided in the water bath to

accommodate a thermostatically controlled heating unit

 

and a motor-driven stirring propeller.

Measurement of Oxygen Consumption

Nine ml of distilled water were placed in each

 

compensating flask. Tissues prepared as described were
placed in the Warburg vessels, each of which contained
2.0 ml of Krebs-Ringer-Phosphate incubation media with a
concentration of 100 mg/100 ml glucose (Appendix 1). A
filter paper wick and 0.2 ml of 10% KOH were added to the
center well of the reaction vessel. The side arm stOppers
of the Warburg vessels were inserted and turned to allow
passage of gas from the vessel. All vessels then were
attached to the respirometer. Preparation of the respir-
ometer to receive the vessels consisted of Opening the
manifold stOpcock to atmospheric pressure, positioning
the three-way stOpcock to short-circuit the manometer,
and opening each respirometer unit to the common manifold.
The flasks were then gassed simultaneously with
100% oxygen for approximately three minutes. Oxygen

entered through a rubber hose from a tank to the stopcock

._...._____.., ,V.W,"‘I“- {.33 C156? 0 *-

 

45

end of the common manifold, and gas left via the open side
arm of each vessel. After the gassing procedure, the side
arm of each reaction vessel was closed and the entire
apparatus was placed in the constant temperature bath.
The polyethylene tubing was connected to the syringe
microburet on one end, and the shaking rod fixed to the
bench at the other.

The tissues and media were allowed to equilibrate
for 30 minutes while shaking at a frequency of 100-110
cycles per minute and a stroke of 2.8 cm. This time and
frequency promoted adequate oxygenation of the media so

that oxygen diffusion could be excluded as the limiting

factor in any results (Umbreit, Burris and Stouffer, 1964).

To start the incubation following equilibration,
stOpcocks were first closed to separate the units from
the manifold, and then the three-way stOpcocks were turned
so the manometers were connected to the reaction vessels
and compensating flasks. Time, temperature, and baro-
metric pressure were noted at that time. The manifold
remained open to atmospheric pressure.

At the end of the 24 hour incubation, the shaker
*was stopped, the syringe was filled to the capacity of
the micrometer, and the manifold was closed from the at-
mOSPhere. The initial setting on the micrometer was

recorded, then the first unit was opened to the manifold.

 

 

a!"

 

46

The manometer fluid was restored to the zero point by
manipulating the micrometer. The new micrometer reading
was recorded. A change in gas volume was measured
directly by the difference in micrometer readings. The
first unit was closed and measurements were made
successively on the remaining units as described. The
syringe was recharged with oxygen from the manifold and
the above cycle repeated as often as necessary.

Complete compensation for pressure changes due to
temperature and barometric pressure variations was
accomplished by using the first unit as a thermobarometer
containing media only. Adjustment of this unit as de-
scribed above will set the pressure in the manifold equal
to the pressure in the compensating flasks; thus, the
readings are automatically corrected for the remaining
units. The volume of gas consumed by the tissue in each
unit was corrected to STP using 13 C and the original
barometric pressure as the initial conditions.

Before removal of the apparatus from the water
bath, the three-way stOpcocks were always turned so the»
.manometers were eliminated from the circuit to prevent

(expulsion of the manometer fluid.

Comparison of Results with
Le Microrespirometer
Oxygen consumption of rat liver slices in the

c<3>11«Structed microrespirometer was compared with values

 

. "‘i‘.'i".4-— _

 

 

 

 

Cl)

 

47

for liver slices measured by Reineke (1961) when he used
his apparatus and the Warburg microrespirometer simul-
taneously.

Rats were killed by a sharp blow to the head.
The livers were quickly removed, and rinsed in a chilled

Ringer-PhOSphate buffer. Liver slices were cut 0.5 mm

‘1! N 2| no.”

thick with a Stadie-Riggs hand microtome and replaced in

the buffer. Pieces of tissue (80-140 mg) were blotted,

- 'A‘J‘ .'-— it".

rapidly weighed, and placed in reaction vessels contain-

 

Ii.

ing Ringer-Phosphate buffer with glucose. Triplicate
samples from each liver were dried for 12 hours at 100 C
to determine per cent dry weight. Eight samples of liver
tissue from each of three rats were run, totaling 24
determinations. Measurements were made at 15 minute in-
tervals over a period of 60 minutes. The temperature of
the apparatus was controlled at 37 C. The Q02 values
compare well with those obtained by Reineke (1961)

(Appendix 3).

Analysis of Media

Duplicate aliquots of Krebs-Ringer-Phosphate
buffer were taken for glucose determination prior to in-
czubation. After completion of the incubation period,
Santples were again taken from each of the vessels to be
analyzed for glucose. The Glucostat enzymatic micro-

method (Worthington Biochemical Corp., Freehold, N.J.)

 

 

wi t1

tior

the

and

48

with glucose oxidase was used for all glucose determina—
tions.
Lactic acid was determined at the completion of

the experiment by the colorimetric micromethod of Barker

and Summerson (1941).

 

 

an: 1!
i

 

meas

glu:
len
con

48

le

SE

121'

C)

 

RESULTS

General Aspects of Metabplism of Corneal
and Lenticular Tissues

Viability of tissues in-vitro was demonstrated by
measuring oxygen consumption, lactic acid production and
glucose utilization. The in-vitro oxygen consumption of
lenses and corneas of rainbow trout eyes was found to
continue at a constant rate over a period of as long as
48 hours (Appendix 3). In studies of CO2 production
tissues were removed and placed in separate vials which
contained the labeled precursor. Tissues from one eye
served as a control for tissues taken from the contra-
lateral eye and no statistical difference in rate of

14

C 0 production was noted (Appendix 3).

2
Data presented below are for corneal tissue and
lenses only and in all cases paired (right and left eye)

samples were run.

Production of C1402 from 2-C14-pyruvate:

Effect of Inhibitors

The inhibitors were added to the media bathing
the tissues and the data are presented in Table 1. Sodium

cyanide (lo-3M) caused nearly complete inhibition of

49

 

 

50

c140

2
10-3M potassium iodoacetate had no effect.

production by both lenses and corneas whereas

TABLE 1. Effect of inhibitors on production of C140

from 2-Cl4-pyruvate by ocular tissues
of trout at 13 C

2

 

 

 

 

C1402 (dpm/100 mg wet tissue)
Tissue No inhibitor Inhibitor
’ -3 a b
NaCN(lO m) 3989.4:1332.9(5) 5.7:2.1(5)
Lens _3 c
KIAA(10 M) 3660.1i996.3(5) 3927.6i1365.8(5)
NaCN(10-3M) 31,974i14,124(5) 17.8i12.3(5)b
Cornea _3
KIAA(10 M) 29,455ill,477(5) 31,886:5,661(5)

 

 

 

éMean i standard error (observations)
Significant at the p = less than 0.05 level
otassium iodoacetate

Formation of Labeled CO2 from

G—i-cl4 and G-6-cl4

 

 

Untreated Tissues

 

Data presented in Table 2 show that the CO2

production by the cOrnea is about 20 times greater than

that by lenses. Both tissues produced over twice as

much labeled CO2 from the G-l—C14 as from G-6-C14. These

differences were statistically different at the p = 0.01

level.

 

 

 

_,

  

51

2 from G-l-C14 and G-6-C14 by

untreated tissues in PBS at 13 C

TABLE 2.--Comparison of C140

 

 

14

 

 

C 02 (dpm/100 mg wet tissue)
14
. 14 14 G-l-C
Tissue G-l-C G-6—C
G-6-cIz
Lens 154.3:17.9(18)a 66.3:7.4(16) 2.33
Cornea 3589.8:77.2(18) 1295.9:17.1(17) 2.77

 

 

 

 

a .
Mean i standard error (observations)

Effect of Cyanide

 

In this series, eXperiments were performed using

G-l-Cl4 and G-6-Cl4 separately as the original source of

C1402. Sodium cyanide (10-3M) caused a complete suppres-

sion of C1402 formation from G-6-C14 by both corneas and

lenses. In contrast, when G-l-C14 served as the substrate,
lesser but significant (p = 0.03) reductions of 70% and

60% occurred in the lenses and corneas respectively.

None of the tissues used exhibited any visible change in

physical appearance or loss of transparency during the

course (4 hours) of these experiments.

.l .. .-nQ'¢-." .“

l-o

 

 

TABLE 3.--Effect of NaCN (10-3M) on C140

 

14

G-l-C and G-6-C

52

14

2 production from
by tissues in PBS at 13 C

 

 

 

 

C1402 (dpm/100 mg wet tissue)
Tissue Substrate No inhibitor inhibitor figflgfiH—x1oo
14 a
Lens G-l-C 94.8i12.6(5) 28.6i4.5(5) 30.1
Cornea G-l-Cl4 3250.1i39l.7(5) 1294.1i187.4(5) 39.8
Lens c-s-cl4 71.3i8.0(5) o.o:o.o(5) 0.0
Cornea G-6-C14 1008.1i99.1(5) 1.6i0.7(5) 0.1

 

 

 

 

 

 

a .
Mean i standard error (observations)

Effect of Anoxia

The paired eXperimental design was again used.

One incubation chamber received the tissue and media

which had been previously deoxygenated by bubbling N

through it.

The chamber was then gassed with N

2I

2
The

other chamber containing tissue and oxygenated media was

flushed with 100% oxygen prior to being sealed for incu-

bation.

reduced C O
cornea by 75% and 67% respectively.

from G-6-C

A lack of oxygen significantly (p = 0.03)

14
2

14

production from G-l-C

14

in the lens and

14

Production of C 02

by the lens showed a 90% decrease under

anoxic conditions, but formation by the cornea was

 

 

 

 

 

‘3

 

f r
a fl

    

53

reduced only 52% (Table 4). All tissues were transparent

at the end of the four hour incubation period.

TABLE 4.--Effect of anoxia on C140 production from

2
G-i-cl4 and G-6-cl4

 

 

 

 

 

 

1 7
C 402 (dpm/100 mg wet tissue) '1
Tissue Substrate Aerobic _ Anaerobic A%-:-§£LX100
14 a 1
Lens G-l-C 238.9i56.9(5) 57.7il6.9(5) 24.1
Cornea c-i-c14 4979.5:343.1(5) 2121.1:139.7(5) 42.6
Lens G-6-cl4 75.9:21.2(5) 7.344.4(5) 9.6
Cornea G-6-cl4 1361.7:189.0(5) 652.0:120.8(5) 47.8

 

 

 

 

 

a .
Mean i standard error (observations)

Effect of Anpimycin A
In—vivo and In-Vitro

 

In this experiment 5 ug of Antimycin A were in-
jected in-vivo into the anterior chamber of one eye.
After an interval, tissues were removed and C1402 forma-
tion from labeled precursors was measured. Antimycin A
was added directly to the media for another part of the
experiment to compare its in-vitro effect on the C1402

produced.

 

 

F '7‘“

 

 

  

54

 

Antimycin A, when administered either in-vivo or

in-vitro, nearly abolished C1 O

2

14

formation from G-6-C

by both lenticular and corneal tissues (Table 5). When

c-i-cl4

was the precursor, each tissue reduced C O

14
2

production to the same extent, independent of the method

of administration of the inhibitor.

Lenses subjected to

Antimycin A reduced C1402 production by 19% and corneas

by approximately 46%.

TABLE 5.--Effect of Antimycin A in-vivo and in-vitro on

c140

2

yield from G-l-C

and G-6-C

14 14

 

 

14

C O (dpm/100 mg wet tissue)

2

 

G-i-cl4

No inhibitor

Inhibitor

inh

No inH.XI00

 

173.1i33.0(4)b

 

 

Lens in-vitroa 139.6i23.4(4) 80.6
Lens in-vivo° 184.9i35.1(4)b 149.2:23.6(4) 80.6
Cornea in-vitro 3340.3i475.5(4) 1841.7i138.8(4) 55.1
Cornea in-vivo 3131.0i83.3(4) 1665.2i181.8(4) 53.1
G-6-Cl4
Lens in-vitro 61.3:8.9(4) 0.710.2(4) 1.1
Lens in-vivo 54.9:12.9(4) 1.510.3(4) 2.7
Cornea in-vitro 1682.3i123.0(4) 15.0i4.4(4) 0.9
Cornea in-vivo 1367.93302.2(4) 114.9i29.3(4) 8.3

—_

 

 

 

 

a .
Mean 1 standard error (observations)

Antimycin A concentration of 2.6 ug/ml in media

cAntimycin A (5 ug) injected into anterior chamber of the eye

 

 

 

 

55

The addition of 5 ug of Antimycin A to the
anterior chamber of trout eyes resulted in a change of
the physical characteristics which were evident by 48-72
hours after injection. Eyes in treated fish appeared
swollen and a slight loss of pigment was evident in the
iris and the limbus of the corneas. Lenses were opaque
to the extent that they were clearly visible from the
exterior and they showed an approximate 20% gain in
weight. Corneas appeared cloudy and were more affected
at the periphery than in the center. Most unusual was
the presence of a gas bubble in the anterior chamber of
all the fish injected with inhibitor. Contralateral eyes
of the fish which were injected with an equal volume of a
solution similar in content to the experimental solution
but lacking Antimycin A, showed no visible changes for a

period of 72 hours after injection.

Effect of Temperature

 

Experiments contrasting the C1402 production from

G-l-Cl4 and G-6-Cl4 were performed at temperatures from

4-43 C.
With an increase in temperature, formation of

C1402 from G-6-Cl4 by lenses rose rapidly and reached a
maximum at 23 C. Above this temperature there was an
equally rapid fall in C1402 production (Figure 5). With
G-l—Cl4, C1402 production sharply increased between 13

and 23 C, then leveled off to reach a maximum at about

 

 

 

56

33 C (Figure 5). At this temperature about 17 times more
C1402 was formed from G-l-Cl4 than from G-6-Cl4 metabolism.
Lenses retained their transparency after 4 hours at temp-
eratures up to 33 C but clouding occurred in 45 to 60
minutes after the start of incubation at 43 C.

TABLE 6.--Effect of temperature on C1402 production from

G-l-Cl4 and G-6-Cl4 from tissues in PBS at 13 C

 

 

 

 

 

C1402 (dpm/lOO mg wet tissue)
Temp. Lens Cornea
G-i-cl4 G-6-cl4 G-i-cl4 G-6-cl4
4 C 135.3i3l.6a 21.4i7.2 1404.5i234.3 262.0i59.9
13 C 162.4i8.0 47.5il.4 3512.8i102.l 1278.9:833.4
23 C 609.2:38.5 96.0:5.2 9628.4:1046.6 3568.5:503.0
33 C 616.8:79.5 35.6:8.9 4576.2:677.0 666.4:126.l
43 C 559.1:39.5 29.7:10.5 1239.4:232.2 110.9:13.7

 

 

 

 

 

a I
Mean of 4 observations i standard error

The formation of C1402 from G-l-Cl4 by corneas

rose rapidly when temperature was increased and, like
lenses, showed a peak production at 23 C with a sharp
decline above this temperature. The profile is similar
for C1402 formation from G—6-Cl4 but about one-third the

magnitude at 23 C (Figure 5). At temperatures above 23 C,

 

 

"in...

 

.3.
I . .1

..i...‘ up."

 

 

57

Figure 5.—-The effect of changing temperature
on the C1402 formation from G—l-C14 and G—6-Cl4 from

the lens and corneas incubated in PBS.

 

10,000

9,000

8,000

7,000

6,000
5,000
4,000

3,000

C1402 (dpm/100 mg wet tissue)

2,000

1,000

700
600

500
400

300

200

(dpm/lOO mg wet tissue)

2

100

I4

 

58

 

 

 

 

 

 

p Cornea
‘ G-l-Cl4 *3
E
2.5:
. _i l l l I l 1 l
10 20 30 40
Temperature C
P #1 Lens
- T [‘\\\“\f .
' c—1-cl4

 

 

 

Temperature C

Figure 5.

 

 

59

corneas became cloudy and their edges assumed a scalloped

appearance shortly after the incubation period began.

Effect of Capsule Damage

The lens capsule was cut with a sharp scalpel.
As the cut was made, the contents within the lens appeared
to extrude out of, and enlarge the opening as if the
capsule had been under tension. The C1402 production
from five of these lenses was compared to that from in-
tact lenses. Data in Table 7 show no significant change
in C1402 production after the capsule was damaged.

TABLE 7.--Effect of capsule damage on C1402 production

and Cl4 tissue content of lens incubated in G-l-Cl4

 

 

Activity (dpm/100 mg wet tissue)

 

 

Lens Intact Damaged
14 a
c 02 i92.3:29.0(5) 207.2:25.2(5)
Tissue Cl4 9,843147l(5) 10,538i527(5)

 

 

 

a .
Mean i standard error (observations)

Effect of Inhibitors on Tissue Cl4 Activity
After Incubation with G-l-C14
In experiments to determine the effects of
Various inhibitors on the presence of Cl4 activity in

thfii tissues after incubation, the tissues were solubilized

 

 

 

in 1.0 ml of Hyamine.

60

a liquid scintillation system.

The activity was then counted in

14 . . . . .
TABLE 8.--The C actiVity in ocular tissue after incuba-

tion with G-l-C14

and various inhibitors

 

 

Tissue C14

activity

(dpm/100 mg wet wt.)

 

Lens

No inhibitor

Inhibitor

 

NaCN (10-3M)

12,108i708(4)a

11,061i664(4)

 

 

Antimycin A (2.6 ug/ml) 12,613i510(4) 9,714:296(4)

N2 atmosphere 13,810i321(4) 12,255i564(4)
Cornea

NaCN (10-3M) 50,383i1237(4) 44,708:476(4)

Antimycin A (2.6 ug/ml) 41,086i2260(4) 42,642i2761(4)

N2 atmosphere 53,738i965(4) 52,462i1401(4)

 

 

 

a I
Mean i standard error (observations)

No significant difference in activity was seen

'when the radioactivity of the tissue subjected to NaCN

(10-3M), Antimycin A (2.6 ug/ml), or an N2 environment,

‘Mas compared to the activity of the untreated controls

(Table 8). The activity present in the corneal tissue

was approximately four times that in the ~1ens when cal-

culated on a per unit volume basis.

tug...

 

 

 

61

Effect 9f Inhibitors 9n the C1

in Tissues After Incubation with

2—C14-pyruvate

Tissues were incubated with 2-C1

activity contained in each tissue at the end of the ex-

periment was determined.

TABLE 9.--Effect of inhibitors on C14
tissue after incubation with 2-C

4 Activity

activity in ocular
4-pyruvate

4--pyruvate with

and without the presence of inhibitors and the C14

 

 

 

Tissue C14 activity (dpm/100 mg wet tissue)

 

 

-£2§L——X100

 

 

 

Lens No inhibitor Inhibitor No inh.
NaCN (10-3M) 47,363i6197(4)a 21,051i1769(4) 44.4b
KIAA (10'3M) 41,9951357st4) 38,043i4362(4) 90.5

Cornea
NaCN (10'3M) 121,751:23,559(4) 57,943i4358(4) 47.51”

83.1

KIAA (10'3M) 92,746s7061(4) 77,532i6629(4)

 

 

 

¥

 

aMean 1 standard error (observations)
:bSignificant at the p = 0.03 level

The addition of cyanide to either the lens or

(Harnea caused a significant reduction in tissue radio-

aCtivity to approximately one-half of the amount present

111 the contralateral tissue. No significant difference

 

 

 

62

was apparent when potassium iodoacetate (10-3M) was
added to the media. The cornea without inhibitor

contained roughly 2.5 times more C14 activity than the

lens.

 

 

-lt-§'m.5£~~

 

 

DISCUSSION

Biochemical studies reported here have produced
data to show that oxidation of glucose to CO2 by the
citric acid cycle and via the hexosemonophosphate (HMP)
shunt occurs in lenticular and corneal tissues of rainbow

trout. Potassium iodoacetate, an inhibitor of glycolysis,

 

had no effect on CO2 production from pyruvate. Production
of CO2 from glucose was blocked by known inhibitors of the
electron transport chain (ETC), which is necessary for the
operation of the citric acid cycle.

It is established that the citric acid cycle is
operational in lenses of mammals. Recently Fonner,
Hoffert and Fromm (1969) reported that (based on histo—
chemical data) the presence of the citric acid cycle in
ocular tissues of rainbow trout was equivocal. The
presence of intermediates of the cycle in a tissue is not
unequivocal proof that the cycle is operating in the
tissue since many of the enzymes identified and substrates
IJsed are involved in other known cellular reactions.

Using labeled glucose (G-6-Cl4) as a substrate,
it: was found that the formation of labeled CO2 by both

leIlses and corneas was inhibited when NaCN, Antimycin A

63

 

64

or anoxia were used. Many workers have found that
cyanide severely reduces lens respiration (Herrmann and
Moses, 1945; Ely and Robbie, 1950; Hockwin, Kleifeld and
Arena, 1956), but Kinoshita (1955) reported only 35% in-
hibition of glucose oxidation.

For illustrative purposes, calculations were made E‘l
using specific activities. The amount of C1402 produced

from 2-Cl4-pyruvate indicated that about 0.06 pg pyruvate/

 

100 mg wet tissue was oxidized to CO by the lens and

2
roughly 0.46 ug/100 mg tissue by corneas. A ratio of the

amount of C1402 produced to the amount of 2-C14-pyruvate

 

present in the tissue indicates that the cornea is far
more active in the oxidation of pyruvate than is the lens
of rainbow trout. Similar results have been reported for
mammalian corneas by Kuhlman and Resnik (1959) who found
that when excess lactate was added to the incubation
media as an unlabeled competitor, the CO2 yield from
G-6-Cl4 by mammalian corneas drOpped some 86%. From
their experiments with labeled lactate they concluded
that the efficiency of lactate oxidation was comparable
to that for glucose.

14

When trout corneas were incubated with G-6-C

Hinder anaerobic conditions the production of C140 was

2
Ortly'reduced to about 48% of that which occurred aerob-

ically. If the ETC were truly ineffective due to oxygen

 

65

lack, then an alternate pathway for the oxidation of the
6-Cl4 atom of radio—glucose must exist in the cornea..
It is possible that all oxygen was not removed from the
media and that the tissue contained some oxygen at the
start of the experiment; hence, complete inhibition was

not evident under the "anaerobic conditions" as described.

 

Both cyanide and Antimycin A did; however, completely in—

hibit the ETC and production of C1402.

The metabolic fate of the carbon-l and carbon-6

 

atoms differs depending on how glucose is metabolized. Egj
If a tissue metabolized glucose via the glycolysis—citric

acid cycle exclusively, carbon atoms 1 and 6 of glucose

become the methyl carbon of pyruvate and the complete

oxidation of pyruvate would result in these atoms

appearing as CO2 in equal amounts. The HMP shunt, on

the other hand, preferentially removes carbon 1 of

glucose to form C02.

Based on the above principle, Kinoshita (1955)
and Kinoshita and Wachtl (1958) incubated mammalian
lenses with G-l-Cl4 and G-6-C14 and measured the relative
contributions of the two metabolic pathways. They found
'that the C-1 atom of the glucose molecule was oxidized to

1
<3 4O at 41 times the rate of oxidation of the C-6 atom,

2
arna that some 85% of the total CO2 produced is via the
Hm“? shunt. If glucose had been metabolized by the

gJ-Ycolytic-citric acid cycle exclusively, then the ratio

 

66

would have been 1 instead of 41. With trout tissues it

was found that those incubated with G-l-Cl4 yielded more

C1402 in all cases than those incubated with G-6-Cl4.
Ratios of C-l-COZ/C-G-CO2 were 2.33 for the lens and 2.77
for the corneas which, when compared with data from
mammals, indicates that the HMP shunt is much less active
in fish than in mammalian ocular tissues.

By making some additional calculations using data

presented in Table 2 a more quantitative relationship

between activities of the HMP shunt and the citric acid

 

cycle in trout lens can be shown. The 66.3 dpm recovered
from G-6-Cl4 represents the amount of glucose oxidized to
CO2 via the citric acid cycle. Since there are six car-

bon atoms in glucose, the total CO production via the

2
citric acid cycle would be 6 X 66.3 or 397.8 dpm/100 mg
wet tissue. The 154.3 dpm listed as coming from G-l-Cl4
includes labeled CO2 produced by both the HMP shunt and
the citric acid cycle; hence, the total amount of CO2
produced by the HMP shunt is represented by 154.3 - 66.3
or 88.0 dpm/100 mg wet tissue. Therefore, in the trout
lenses the C02 produced via the HMP shunt represents about
1/5 (or 22%) of the total CO2 produced by this tissue.
Similar calculations for trout corneas show the HMP shunt

activity accounts for about 1/3 of the total CO2 produced

by this tissue. These results differ from data for

 

 

-w;I-+

   

 

‘5‘: I

 

67

comparable mammalian tissues which indicates that the HMP
shunt accounts for about six times more CO2 than the
citric acid cycle or about 85% of the total produced.

All evaluations of the activity of the two path-
ways were made without consideration for the effect of
recycling of compounds through the HMP shunt with the
subsequent rearrangement of isotopic carbon atoms. Katz
and Wood (1960) estimated the amount of randomization of
C14 which will occur in the glucose-6-phosphate at steady
state when glucose-C14 is metabolized via a complete
pentose cycle. They have shown that the extent of ran-
domization of Cl4 is proportional to the fraction of the
total metabolism of glucose-C14 that proceeds by the
pentose cycle. The level of activity of the HMP shunt
appears to be low in the lens and cornea of rainbow trout;
therefore, recycling of the HMP shunt would be of minor
significance.

Data on the effect of temperature indicates that
the lens responds to increases in temperature by increas-

production from G-l-Cl4 to a much greater extent

2
than from G-6-Cl4. In other words, the HMP shunt activity

ing C0

shows a greater response to increased temperature than

citric acid cycle activity.~ At the preferred temperature

of trout, which is about 13 C, C140 production from the

2
HMP shunt is about 3 times greater than citric acid cycle

14

C 02 production. At 33 C it is 17 times greater. This

 

 

 

   

68

figure is somewhat comparable to the ratio of 41 reported

above for mammalian corneas at 37 C. Above 33 C the HMP
shunt activity in fish lenses declines and at these
temperatures they become Opaque during incubation.
Activity of the HMP shunt appears to be quite sensitive
to temperature and as temperature goes up, it may become

the prime contributor of the energy required to maintain

the normal transparency of the lens. It is possible that

above 33 C, the HMP shunt cannot produce enough energy to

maintain lens transparency.

The cornea responds to temperature changes some-
what differently than the lens. As previously stated,
the citric acid cycle is initially more active in the
cornea and it reSponds to increased temperature by
increasing activity up to 23 C. At this temperature the
ratio (C-l-COz/C-G-COZ) remains at approximately 3. As

the temperature is increased from 23 C to 43 C the 01402

yield from G-l-C14 falls rapidly and above 23 C changes
in the Opacity along with other physical changes occur
in the cornea. Perhaps transparency could be maintained
at elevated temperatures if the HMP shunt could respond
with increased activity above 23 C. One is tempted to
speculate that at temperatures normally encountered the
trout cornea depends primarily on the citric acid cycle

for energy production, and that the added increment of

energy necessary to maintain transparency at high

 

 

 

 

   

69

temperatures is dependent upon increased HMP shunt
activity. Corneal HMP shunt activity appears more limited
in its response to increased temperature than is the HMP
shunt of trout lenticular tissue.

The differences noted between trout and mammalian
eyes may represent significant adaptations permitting
normal function under very different conditions. Trout
eyes seem to depend mainly on the activity of the citric
acid cycle and very little on the HMP shunt for produc-
tion of usable energy. They are able to maintain normal
transparency in lens and cornea at temperatures approach-
ing 0 C. Clouding of these normally transParent structures
at high temperature coincides with decreases in HMP shunt
activity. In mammalian ocular tissues HMP shunt activity
appears to be the prime provider of usable energy and may
represent a metabolic adaptation permitting maintenance
of transparency at higher temperatures. Mammalian ocular
tissues become Opaque at lower temperatures (Harris,
Gehrsitz, and Nordquist, 1953), but the way various
metabolic reactions are involved is not known.

The constant temperature chamber used for the
temperature studies had a limited working space and all
preparation of tissues and media had to be carried out at
room temperature (23 C). This may have introduced a
source of error into the data from experiments run at

temperatures different than room temperatures because

 

 

 

 

70

there was no equilibration of the incubation vessel to
the experimental temperature level before the experimenta-
tion period began. The experimental period was started
when the tissue was placed in the media at room tempera-
ture and the vessels were sealed. The metabolic rate of
the tissue during the time of changing temperature would
be different from the rate of metabolism at the desired
temperature. In the experiment at 43 C, a temperature
which is known to denature some protein enzymes, a rate
of metabolism existed during the period of temperature
change which might not have been present at all if
equilibration of the tissue to 43 C had occurred prior
to the start of the experimental period.

Based on the production of C1402 it was found
that disruption of the lens capsule did not alter the
metabolism of this tissue. The technique used to produce
lesions was admittedly crude and the extent of lenticular
damage was variable and difficult to ascertain. The fact
that no change was observed under these conditions suggests
that the capsule of the trout lens is not an impenetrable
barrier against the inward diffusion of glucose. Ely
(1949) has reported that the isolated capsule and nucleus
of bovine lens has a negligible respiration; however, he
also found that rupturing of the capsule increased the

oxygen uptake 2-4 times. Rae (1968) showed that damage

 

 

I'
‘0
'.
'7‘
i

 

 

 

"‘ "' '.' i"?- "7 ""J""'"¥."f§fifi.w—qy—r_a _fi__ ‘.‘“.-. __ ,

   

71

 

to the capsule caused a nearly 50% reduction of the
potential difference of the lens of rainbow trout. He
contends that the capsule is a limiting membrane to ion
diffusion.

Glucose that entered the tissue was determined by
counting the Cl4 activity of the solubilized tissue after
completion of the experimental period. At that time the
C14 may have been incorporated into metabolic intermedi-
ates, but it was assumed that the radiolabeled carbon
entered as part of glucose molecules. Calculations made
from the specific activity of the media provided informa-
tion on the amount of glucose that entered the tissue.

The concentration of glucose in the media was
90.8 ug/ul. The glucose in lenses weighing about 100 mg
was 21.8 mg but the volume of tissue in which this was
contained is subject to discussion. If one assumes that
glucose is evenly distributed throughout the lens, then
a 100 pl volume (assuming 1 mg = 1 pl) lens would have an
internal concentration of 21.8 ug/lOO ul. A diffusion
gradient would then exist which would favor entry of
glucose into the tissue. However, the teleost lens
differs from its mammalian counterpart in that it is a
Sphere, does not change shape for accommodation, and has
a greater percentage dry weight. The increased dry weight
is manifest in a nucleus which is fibrous, extremely hard,

and resistant to digestion by Hyamine at 60 C for 18 hours.

 

 

‘.

   

72

 

Beads of undissolved lenticular nuclei, differing in
size, remained in the scintillation vial after 18 hours
digestion but their presence did not affect the counts.
This leads one to believe that the beads did not contain
radioactive glucose so glucose could not have penetrated
the lens to this depth in 5 hours.

If one assumes that glucose entrance into the
lens occurs solely by a passive diffusion process, a
volume within the lens can be calculated in which 21.8 ug
of glucose is distributed to obtain an equilibrium con-
centration with the media. This volume was found to be
24.0 ul. Further assuming that this volume is evenly
distributed over the surface of the lens, then its thick-
ness would be some 230 microns and would represent the
depth of penetration of passively diffusing glucose.
Rae (1968) showed that the potential difference of the
lens which is dependent on the diffusion of ions into the
lens, existed to a depth of only about 100 microns from
the surface.

Neither lesioning the capsule nor the addition
of inhibitors affect the entry of glucose into the tissue.
This differs from the results of Harris, Hanchild, and
Nordquist (1955) who showed that the accumulation of
glucose within the mammalian lens could be altered by

various enzyme inhibitors. Perhaps the energy dependent

 

 

 

 

73

system for uptake of glucose is an adaptation by the
mammalian lens to meet the greater energy demands

necessary for maintenance of transparency.

 

The concentration of glucose in trout corneas
was found to be equal to that of the incubation media.
The anterior surface of the trout cornea has been shown
to be impermeable to ions and very likely to glucose as
well (Edelhauser, Hoffert, and Fromm, 1966). The endo-

thelium has been described as incomplete and poorly

 

develOped in comparison to mammalian lenses (Hoffert and

Fromm, 1965). Therefore, equilibrium concentration of

  

glucose in the cornea could be achieved by entry through
both the endothelium and the limbal area where the insci-
sion was made. The use of inhibitors failed to alter the
rate of entry into the cornea.

When trout tissues were incubated in a medium

which contained pyruvate at a concentration of 0.75 ug/

 

100 ml, lenses had mean concentrations of pyruvate of

‘ 5' "it“: *V‘z‘9'“*m‘rms‘mmwvxwflrflmm; "

0.68 ug/100 mg wet tissue whereas corneas accumulated
pyruvate to a concentration of 1.75 ug/lOO mg. The fact

3M NaCN the amount of

that after poisoning with 10-
radioactivity in both lenses and corneas was about half

the concentration attained in non-poisoned tissues suggests
the presence of an energy dependent mechanism for pyruvate

transport in both tissues. Cyanide could alter the amount

     

74

0f pyruvate entering a tissue by inhibiting a membrane
transport system that is directly responsible for transfer
of pyruvate or cyanide could cause a buildup of endogenous
unlabeled pyruvate thereby reducing a membrane diffusion
gradient causing less labeled pyruvate to enter by
passive diffusion. The low concentration of pyruvate in
the lens may result from pyruvate being confined to a
limited lenticular volume as suggested above for glucose.
Also, since cyanide blocks the ETC, pyruvate could enter
the tissue at a normal rate and then be converted into
lactic acid. The trout lens does contain lactic dehydro-
genase (LDH) and any lactate formed would be a freely
diffusable substance which could leave the lens carrying
the c14 label with it.

In summary, glucose appears to enter the trout
lens and cornea as a result of a different mechanism than
is postulated for similar mammalian tissues. Neither
lesioning the lens nor use of specific inhibitors for the
TCA cycle on the lens or cornea changes the rate of entry,
whereas, specific inhibitors used with mammalian tissues
show a marked reduction of glucose entry. Inside the
tissue, glucose is oxidized primarily through reactions
of the TCA cycle at the environmental temperatures of
rainbow trout. As the temperature rises, the HMP shunt

responds in both types of tissues with a greater increase

 

 

 

.wm-chwu-IWWMMW , '

 

 

 

75

in activity than the TCA cycle. This increase in activity
lasted to 23 C in the cornea but persisted to 33 C in the
lens, the respective temperatures where the tissues

became opaque .

 

 

 

 

CONCLUSIONS

The TCA cycle is present and actively oxidizes
pyruvate to CO2 in both the lens and cornea, but the
activity of the cycle is greater in the cornea than
the lens.

The HMP shunt is present, but accounts for only about

1/5 of total CO production by the lens, and 1/3 of

2

total CO production by the cornea.

2
The activity of the TCA cycle in both the lens and
cornea increases with rising temperatures to 33 C,
then declines to 43 C; however, the cornea responds
with a greater magnitude of increase than does the
lens.

The HMP shunt is able to increase activity with
rising temperatures to 23 C in the cornea before a
decrease occurred, while shunt activity increased
with temperature to 33 C in the lens.

Entrance of glucose into the lens or cornea is not
dependent on energy production of the TCA cycle.
Pyruvate enters the lens and cornea aided by an

active TCA cycle.

76

 

 

.. <7 '.- ..—

 

 

 

77

7. iIn-vivo treatment with Antimycin A which causes
opacities in both the lens and cornea also results
in substantial reduction of energy produced from the

series of reactions for the breakdown of glucose.

 

 

 

LITERATURE CITED

Allison, L. N. 1963. Cataract in hatchery lake trout.
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Barker, S. B., and W. H. Summerson. 1941. The color-
metric determination of lactic acid in biological
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Edelhauser, H. F., J. R. Hoffert, and P. O. Fromm. 1966.
Water permeability of normal and pathological
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Ely, L. O. 1949. Metabolism of the crystalline lens.
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Ely, L. O. 1951. The oxidation of lactate, pyruvate,
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Ely, L. O. and W. A. Robbie. 1950. The cyanide sensit-
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Fairbanks, M., 1968. Characterization of the normal
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graphic electro e. Thesis, Michigan State
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Field, J., E. G. Tainter, A. W. Martin, and H. S. Belding.
1937. Studies on the oxygen consumption of the
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Fisher, F. P. 1931. Ernahrung und stoffwechsel der
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78

 

 

 

79

Foruier, D. B., J. R. Hoffert, and P. O. Fromm. 1969.
The influences of lactic dehydrogenase on the
metabolic pathways of glucose in the ocular
tissues of rainbow trout (Salmo airdneri).
Comp. Biochem. and Physiol., Accepte or Pub.
Feb., 1969.

Foye, R. E. 1969. Effects of a low-dosage application
of Antimycin A on several species of fish in
Crater Pond, Aroostook County, Maine. Progr.
Fish Cult., 31:216—219.

Giardini, A. and J. R. E. Roberts, 1950. Concentration
of glucose and total chloride in tears. Brit.
J. Ophthal., 34:737—743.

Green, H., C. Bocher, and I. H. Leopold. 1955. Anaerobic
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Haberick, F. J. and R. Dennhardt. 1965. Measurement of
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Harris, J. E., L. B. Gehrsitz, and L. T. Nordquist. 1953.
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Harris, J. E., J. D. Hanschild, and L. T. Nordquist.
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Harris, J. E., J. D. Hanschild, and L. T. Nordquist.
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Heald, K. and M. E. Langham. 1956. Permeability of the
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Herrmann, H. and S. G. Moses. 1945. The cytochrome
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80

Harmann, H., S. G. Moses, and J. S. Friedenwald. 1942.
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Hockwin, 0., O. Kleifeld, and P. Arens. 1956. Einfluss
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81

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82

 

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83

 

Wagemann, A. 1891. Experimentelle untersuchungen uber
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‘.'J.’ ‘.': .

' .. . “M ' o. 'wv»; svawavmgmvua

 

|IILIl,—w§ut

.......a.wa...«.mkmkawBflvfikflwg”awn.....u..........r..c 6...... .3... 595435.. ET 3., . 3... 9%“ 4.3.... .53.. L - 3.. EL.

APPENDICES

 

 

APPENDIX I

INCUBATION SOLUTIONS

 

Composition of Phosphate Buffered Saline (PBS)

 

NaCL 8.00 gm/liter
KCL .20 gm/liter
NaZHPO4(Anhydrous) 1.15 gm/liter
KH2P04H20 .20 gm/liter
CaCL2 .10 gm/liter
MgCL2'6H20 .19 gm/liter

Composition of Krebs-Ringer—Phosphate Solution

Parts Mixed For

 

 

 

    

Constituent Concentration (M) Whole Medium (m1)
NaCL 0.154 232 L
KCL 0.154 8 %
Mgso4 0.154 2 L
CaCL2 0.110 6 i

The mixture is adjusted to pH 7.4 by the addition
of 0.1 N NaOH. To this is added 12 m1 of M/15 phOSphate
buffer, pH 7.4. (This is prepared by mixing 80.8 ml of
M/15 NazHPO4 with 19.2 ml of M/15 KH2P04.) To avoid pre-

cipitation the calcium solution and phosphate buffer were

84

 

 

O

‘u
”a
. .

       

85

IKIt brought together in less than final volume. The
completed solution however was stable.
Glucose was added to this medium in the amount of
0.4 m1 of 5% solution to 19.6 ml of medium. The resulting
medium contains in millimoles per liter approximately the
following: Na+, 14o; K+, 5; Ca++, 2.5; Mg++, 1; c1", 144;
P, 3 and SO --, 1, giving a total ionic strength of 0.158

4
(umbreit, Burris, and Stouffer, 1964).

 

 

 

 

 

 

 

 

APPENDIX II

COUNTING RADIOACTIVITY

Liquid scintillation counting solution I

Naphthalene 100 g
POP 10 g
POPOP 250 mg

Add Dioxane to make 1 liter.

Liquid scintillation counting solution II

Naphthalene 80 g
POP 5 g
POPOP 100 mg

Add Dioxane to make 1 liter.

86

 

 

 

w;.1g,¢c;.xu

T.‘ o~ g .i

 

 

Figure 6.—-Sample channels ratio quench
correction curve for varying degrees of quenching.

 

 

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s.
a
O
.
O
.

 

o\m oeumm mamccmso

m.

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om

om

ov

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om

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OOH

 

 

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89

APPENDIX III

TABLES

TABLE 10.--Comparison of C1402 production from bilateral
ocular tissues using uniformly labeled glucose-C14

 

 

 

C1402 (dpm/100 mg wet tissue)
Right tissue Left tissue
Lens 265.8153.0(6)a 300.514l.5(6)
Cornea 2839.3:523.7(6) 3067.21516.3(6)

 

 

 

 

aMean i standard error (trials)

TABLE 11.--Comparison of QO2 values obtained from con-

structed respirometer with literature
values for liver slices

 

Mean Q02 S.E.

 

Rat 1 Rat 2 Rat 3 Mean

 

Constructed
apparatus 4.63:0.26* 5.71:0.32 3.89i0.3l 4.74:0.37

 

Reineke's

values
Warburg 4.73:0.20
New design 4.8610.19

 

 

a .
Mean t standard error of 8 observations

 

 

 

”VS-M '.' .W. V.

 

90

TABLE 12.--Metabolic data from reSpirometer studies

 

 

 

 

 

Cornea Lens

002 @ 13 C.

(pl/gm wet wt-hr) 52.67i2.63(35)a 11.82i0.95(36)
(pl/gm dry wt-hr) 315.95i15.35(34) 26.20i1.81(36)
Glucose utilization

Aerobic (mg/gm-hr) 0.28:0.02(49) 0.14:0.01(33)
Anaerobic (mg/gm-hr) 0.37:0.08(l3) 0.18:0.03(15)
Lactic acid production

Aerobic (mg/gm-hr) 0.21:0.03(50) 0.3010.03(51)

Anaerobic (mg/gm-hr) 0.84:0.13(15) 0.4710.09(15)
Per cent dry weight 12.73il.48(50) 46.98i0.20(51)

 

 

 

aMean standard error (observations)

 

my: 1.3“" . "T _- . _‘

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