Accumulation of rubidium-86 by the rabbit lens
Bernard
Becker
Rabbit lenses incubated in vitro in potassium-free Tyrode's solution for 4 hours accinnulated
trace amounts of Rb-86 to a concentration averaging 18 times that of the incubation medium.
The uptake of Rb-86 was temperature dependent and required calcium. In the presence of
glucose it was inhibited by iodoacetate, ouabain, or digoxin, but not by dinitrophenol,
fluoroacetate, or anaerobiosis. The accumulation ivas inhibited competitively by potassium
ion, and saturation kinetics with nonlabeled rubidium could be demonstrated. In the absence
of glucose the accumulation was reduced, required oxygen, and was much more sensitive to
cyanide, dinitrophenol, and fluoroacetate.
T,
proximately 1 fie per milliliter of Rb-86. The
Tyrode's solution used had the following composition (milliequivalent per liter): Na+ 149, K>
3.0, Ca++ 3.5, Mg++ 1.0, Cl- 144, H2PO<- 0.4,
HCO:r 12, glucose 5.5. The medium was adjusted
to pH 7.3 to 7.5 by bubbling CO- through the
solution. Before use, the Tyrode's solution was
oxygenated for 5 to 10 minutes. When potassium
or other ions were omitted from the medium, the
sodium chloride concentration was suitably adjusted. So-called "K+-free," "Ca++-free," or
"glucose-free" media merely indicated the omission of these substances from the incubation
solutions, but not their removal or depletion from
the freshly enucleated normal rabbit lenses. All
lenses were examined under a dissecting microscope at the end of incubation. If damaged, the
results were disregarded.
The Rb-86 was obtained from Oak Ridge
National Laboratory and had a half-life of 18.66
days. Incubations were carried out with gentle
shaking in a water bath at 37° C. for 4 hours.
In addition, longer time series were run as well
as incubation at other temperatures. Samples of
the media were removed every hour and counted
in a well-type scintillation counter. At the end
of the incubation, the lenses were blotted and
weighed. In some instances they were dried to
determine the percentage of lens water (average
65 per cent water). In other instances the lenses
were homogenized, proteins precipitated with 10
per cent trichloroacetic acid, and counts of the
supernatant compared with those of the medium.
In view of the excellent agreement of the two
methods, sampling the medium every hour was
. he lens affords an opportunity for the
study of cation transport in an intact organ
in vitro. Most published experiments on
the lens have used the technique of recovery of potassium and sodium concentrations after their distortion by prolonged
refrigeration.1' 2 The present study describes
a simpler, more convenient method for
evaluating cation transport in the lens. It
makes use of the remarkable similarity of
the handling of rubidium and potassium by
cation transport mechanisms.
Methods
Albino rabbits weighing 2 to 3 kilograms were
killed by air embolus. The eyes were enucleated
promptly and opened posteriorly to expose the
lens. The vitreous was carefully dissected free.
The lens was freed by carefully rupturing the
zonules with a lens loupe, and placed in 2 ml.
of a modified Tyrode's solution containing ap-
From the Department of Ophthalmology and the
Oscar Johnson Institute, Washington University
School of Medicine, St. Louis, Mo.
This investigation was supported in part by Research Grants B-1375 and B-621 from the
National Institute of Neurological Diseases and
Blindness, United States Public Health Service,
Bethesda, Md.
502
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Volume 1
Number 4
Accumulation of rubidium-86 by rabbit lens 503
selected, for it provided better estimates of the
progress of incorporation of rubidium from the
medium during the entire experiment. All counts
were expressed as counts per kilogram of lens
water, and the ratios T/M of counts in the tissue
(T) to that in the medium (M) were utilized.
Results
1. Accumulation. The accumulation of
rubidium in the lens remained approximately linear for the initial 4 to 6 hours.
In the "potassium-free" medium, a T/M
ratio averaging 18 was found at 4 hours
for 300 control lenses used in the experiments (Fig. 1). In the Tyrode's solution
containing 3 mEq. K+ per liter, the T/M
ratio averaged 12 at 4 hours. Lenses from
different animals varied in T/M ratio from
9 to 30 in a K+-free medium. Since the
lenses of the two eyes accumulated Rb-86
at remarkably similar rates, it was found
best in evaluating medium requirements,
inhibitors, temperature effects, etc., to use
one lens as a control for the opposite lens
of the same animal. The experimental lens
accumulation could then be expressed as
a fraction of the control (Fig. 1).
2. Requirements in the media. A comparison of TC199 with the Tyrode's solution with comparable amounts of K+ failed
to show any difference in accumulation at
4 hours.
A marked decrease in accumulation was
noted when the medium was made glucosefree. This was more marked when the
medium was glucose- and potassium-free
than when potassium was present. In the
glucose- and potassium-free medium, T/M
ratios were reduced by approximately 40
per cent. It required concentrations of glucose in excess of 3.0 mM. to obtain maximum uptake of Rb-86. Galactose could
replace glucose in the medium and restore
normal T/M ratios, but only in concentrations greater than 15 mM. Accumulation
was not altered by the absence of oxygen,
provided that glucose was present in the
medium. In the absence of glucose and
oxygen, accumulation was markedly reduced.
In a calcium- and potassium-free
Trace Rb+ - K+free
0.85mM.Rbf-K+free
Trace Rb*-3m.MK*
1.7mM. Rb+- Kffree
3.3mAiRb+- K*free
6.7mMRbf-/<'free
16,7 mM.Rb+-K+free
60
120 180 240
T IME ( mins.)
Fig. 1. The time course of the accumulation of
Rb-86 by the rabbit lens.
(T/M) o , Ratio of counts in tissue water to those
in the medium for the lens incubated in potassium-free Tyrode's solution with trace amounts of
Rb-86.
(T/M)c, Similar ratio for the contralateral lens
of the same animal subjected to the various concentrations of Rb+ and K+ indicated. Note that
accumulation is linear with time and that accumulation decreases with increasing concentrations of K+ or Rb+.
medium, the accumulation was reduced to
less than 20 per cent of the calcium-containing control. The addition to such a
calcium-free medium of as little as 5 ;u,g
per milliliter of calcium chloride (CaCL)
restored the T/M ratio to that of the control lens. Adding larger amounts of CaCL
failed to alter the T/M ratio.
Variations of pH between 6.5 and 8.0
failed to alter accumulation of Rb-86 in
a potassium-free medium. Values below
6.5 and above 8.0 led to decreased accumulation.
Accumulation in potassium-free medium
was not influenced by the addition of
Pitressin (0.2 units per milliliter), insulin
(1 unit per milliliter), glycine (2 mg. per
milliliter), desoxycorticosterone (0.5 mg.
per milliliter), acetazolamide (3 mg. per
milliliter), 1-alanine (2 mg. per milliliter),
sodium lactate (1.7 mg. per milliliter),
choline (0.5 mg. per milliliter), or sodium
ascorbate (0.2 mg. per milliliter).
3. Temperature dependence. At 0° C.
the accumulation of rubidium in a potassium-free medium was reduced to 5 to 10
per cent of the 37° C. value. The lens in-
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Investigative Ophthalmology
Attgust 1962
504 Becker
f 40
Tyrodes(K+-free)
5 30
*20
io
0>
6
8
10
12
S(Conc.RbCI mM)
14
16
Fig. 2. Variation with concentration of Rb+ in
the medium (S) of the 4 hour accumulation of
Rb-86 in the rabbit lens (v). The upper curve
(open circles) represents the data for potassiumfree Tyrode's solution. The lower curve (solid
circles) is for Tyrode's solution containing 3
mM. per liter of K+.
cubated at 15° C. accumulated approximately 25 per cent as much Rb-86 as its
mate at 37° C. At 25° C. the lens
accumulated some 50 per cent of its 37° C.
value. These findings suggested a Qx0 of
approximately 2.
4. Variation of substrate concentration.
With increasing concentration of nonlabeled rubidium in the medium, the ratio
of radioactivity of the lens water to that
of the medium decreased progressively, approaching unity (Fig. 1). This corresponded to an increase in the total amount
of rubidium accumulated by the tissue,
approaching a maximum value of about
10 mmole per kilogram lens water per
hour (Fig. 2). The data of accumulation
at various rubidium concentrations could
be fit approximately to a Lineweaver-Burk
type plot. The apparent Michaelis-Menten
constant, Km (half-saturation concentration) approximated 2.3 mM. Rb+ and the
maximum velocity was estimated at 42
mmole per kilogram lens water per 4
hours (Fig. 3).
5. Variation of potassium concentration.
When Tyrode's solution containing 3 mEq.
K+ per liter was used and the concentration of nonlabeled rubidium increased
progressively in the media, the T/M ratio
again declined to approach unity. This
corresponded to an increasing accumulation
of rubidium in the lens, approaching a
similar maximum to that found in a potassium-free medium. However, at all values
measured, the lens in Tyrode's solution
accumulated less than did the one in potassium-free solution (Fig. 2). Fitting these
data to a Lineweaver-Burk plot, as in
Fig. 3, resulted in another linear plot which
intersected the line for potassium-free
accumulation on the Y axis. The apparent
Michaelis-Menten constant, Km, for incubation in Tyrode's solution with 3 mEq.
K+ per liter approximated 3.8 mM. Rb+.
The Lineweaver-Burk plots for the two
sets of data were compatible with competitive inhibition by potassium of the
accumulation of rubidium by the lens.
By using varying concentrations of potassium and trace amounts of Rb-86, it was
possible to demonstrate that at potassium
concentrations of approximately 4.5 mM.,
the accumulation of trace amounts of
rubidium was reduced by 50 per cent
(Fig. 4). Similar studies with ammonium
ion in a potassium-free medium suggested
that ammonium concentrations of approximately 12 mM. inhibited the uptake of
Rb-86 by 50 per cent.
1.4
•
1.2
Tyrodes (3mMK + )
•
,'
1.0
^-
.08
.06
.04
T - m « 4 2 m mols/k»
.2
.4
.8
1.0
1.2
Fig. 3. Lineweaver-Burk type plots of the data
presented in Fig. 2. The apparent MichaelisMenten constant K,,, for the potassium-free
medium is 2.3 mM. Rb+ with a maximum velocity
vm of 42 mmole per Kg. lens water for 4 hours.
The data with Tyrode's solution containing 3
mM. K+ intersect the first plot on the Y axis,
suggesting competitive inhibition by potassium
of the Rb-86 accumulation.
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Accumulation of rubidium-86 by rabbit lens 505
Volume 1
Number 4
6. Inhibitors. A variety of metabolic and
other inhibitors decreased the capacity of
the lens to accumulate rubidium. The concentration of inhibitors presented in Table
I resulted in approximately 50 per cent
reduction in Rb-86 uptake when compared with that of the contralateral control
lens. In those instances in which no inhibition was obtained, the highest concentration used was indicated in the Table.
The 50 per cent inhibition values were
estimated from linear plots of the reciprocal of the velocity of Rb-86 accumulation versus the inhibitor concentration.
The inhibition concentrations were also
compared for the potassium-free medium
and the potassium- and glucose-free media
(Table I). The accumulation of Rb-86 by
the lens proved to be remarkably sensitive
to inhibition by iodoacetate in low concentrations (10~5 M). Fluoride was equally
effective in both media, but only in concentrations of 2 x 10"2 M, and ouabain
reduced accumulation in either medium
by 50 per cent at concentrations as low as
7 x 10"s M. Remarkable differences in the
two media were found between the effective inhibitory concentrations of cyanide,
dinitrophenol, and fluoroacetate. In the
media containing glucose, dinitrophenol
(2 x lO"3 M) or fluoroacetate (1 x 10"1 M)
were without any inhibitory effect, and
cyanide levels as high as 2 x 10~3 M were
Table I. Inhibition of accumulation of
rubidium-86 by rabbit lens in potassiumfree medium
Inhibitor"
Iodoacetate
Fluoride
Cyanide
Dinitrophenol
Fluoroacetate
Digoxin
Ouabain
Glucose
present
(5.5 mM.)
1 X 10-5
2
2
t2
fl
3
7
x
x
x
x
x
x
10-a
10-3
10-3
10-1
1CK
10-s
Glucose
absent
3 x lO-s
2 x 10--'
1 x lO-o
4 x io-»
4 x 10-2
3 x 10-'
7 x 10-s
"Concentrations of inhibitors are expressed in moles per
liter required to reduce accumulation to 50 per cent of
the control lens.
fNo inhibition up to and including this concentration.
KJ-4.5H1M.
0
3.0 6.0
a o 12.0 15.0
Concentration KCI (mM)
Fig. 4. The reciprocal of the accumulation ratio
plotted as a function of the concentration of
potassium chloride in the medium (4 hours at
37° C ) .
(T/M) o , Ratio of counts in tissue water to those
in the medium for the lens incubated in potassium-free Tyrode's solution with trace amounts
of Rb-86.
(T/M) C) Similar ratio for the contralateral lens
of the same animal subjected to the various
concentrations of K+ indicated. The linear plot
suggests 50 per cent inhibition of accumulation
at 4.5 mM. K+.
required for 50 per cent inhibition. In the
glucose-free medium, on the other hand,
4 x 10"4 M dinitrophenol, 4 x 10"2 M
fluoroacetate, and as little as 1 x 10~G M
sodium cyanide decreased Rb-86 accumulation by approximately 50 per cent.
Discussion
The 4 hour uptake of rubidium by the
lens incubated in a highly simplified
medium provides a convenient and reproducible tool for studying cation transport
in the lens. The evidence for competitive
inhibition by potassium, the ability to
study the early phases of uptake in extremely fresh lenses, and the over-all
similarity to the findings with potassium
and sodium transport1-2 all suggest the
usefulness of Rb-86 for such transport
studies.
It must be re-emphasized that the media
used in this study, which are called "free"
of potassium, glucose, or calcium, are not
truly devoid of these substances since no
effort was made to deplete the lenses.
Therefore, some of the potassium, glucose,
or calcium contained in the lenses may
migrate into the medium and introduce
small errors.
The present study demonstrates many
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506 Becker
features of Rb-86 uptake compatible with
active transport of rubidium into the lens.
The accumulation occurs against a concentration gradient, is temperature dependent, is altered by competitive and
metabolic inhibitors, and demonstrates
saturation with Michaelis-Menten type
kinetics. The fact that the accumulation of
rubidium is so very sensitive to ouabain
and digoxin may be taken as suggestive
evidence for a role of the sodium-potassium-activated membrane ATPase found
in the lens.8' '
The inhibitory effects of omitting Ca++
from the K+-free medium is to be anticipated. However, in contrast to previous
findings,5 as little as 5 /xg CaCl2 per
milliliter is sufficient to restore accumulation of Rb+ to normal, and higher calcium
levels fail to increase accumulation further.
It is interesting that in the presence of
glucose, the accumulation of rubidium is
relatively little affected by dinitrophenol,
cyanide, fluoroacetate, or anoxia, but is
veiy sensitive to iodoacetate. These findings support the suggestion by Kinoshita'of a lack of dependence of cation transport
in the lens on aerobic phases of glucose
metabolism, and a predominant role of
glycolysis. However, the dramatic effect
of such low levels of iodoacetate and the
apparent relative resistance to fluoride inhibition are somewhat surprising. Unfortunately, the fluoride inhibition studies
Investigative Ophthalmology
August 1962
are complicated by the insolubility of calcium fluoride, limiting the availability of
both calcium and fluoride ions in the
media.
When a glucose-free media is used, the
rate of uptake is maintained at 60 per cent
of normal, requires oxygen, and is much
more sensitive to fluoroacetate, dinitrophenol, anoxia, and especially to cyanide.
These findings suggest that the lens is
capable of deriving energy for this transport from aerobic mechanisms.
REFERENCES
1. Harris, J. E., Hauschildt, J. D., and Nordquist, L. T.: Lens metabolism as studied with
the reversible cation shift, II, The effect of
oxygen and glutamic acid, Am. J. Ophth. 38:
(Pt. II) 148, 1954.
2. Kinoshita, J. H., Kern, H. L., and Merola, L.
O.: Factors affecting the cation transport of
calf lens, Biochim. et biophys. acta 47: 458,
1961.
3. Post, R. L., Merritt, C. R., Kinsolving, C. R.,
and Albright, C. D.: Membrane adenosine
triphosphatase as a participant in active transport of sodium and potassium in the human
erythrocyte, J. Biol. Chem. 235: 1796, 1960.
4. Bonting, S. L., Simon, K. A., and Hawkins,
N. M.: Studies on sodium-potassium-activated
adenosine triphosphatase, I. Quantitative distribution in several tissues of the cat, Arch.
Biochem. 95: 416, 1961.
5. Merola, L. O., Kern, H. L., and Kinoshita, J.
H.: The effect of calcium on the cations of
calf lens, A. M. A. Arch. Ophth. 63: 830,
1960.
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