Download Intracellular calcium concentration and calcium transport in

1032
INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / July 1985
Vol. 26
Introcellulor Colcium Concentration ond Colcium
Transport in the Rabbit Lens
Kenneth R. Highrower,* George Duncan, f and Sharon E. Harrison*
Calcium-sensitive microelectrodes have been employed to
determine that the free, intracellular concentration of calcium
in the lens is approximately 30 nM. Additionally, active
extrusion of intracellular calcium has been demonstrated.
Invest Ophthalmol Vis Sci 26:1032-1034, 1985
Calcium ions appear to play several primary roles
in lens physiology. An increase in lens calcium is
accompanied by an increase in light scatter,1 a decrease
in protein synthesis,2 and a destabilization of the lens
cytoplasmic gel.3 The control of calcium movements
by active and passive mechanisms are, however,
difficult to investigate by conventional radiotracer
methods partly because a large fraction of the total
calcium appears to be bound 4 and partly because of
the extremely slow penetration of 45Ca into the lens.5
In the present study, we employ calcium-sensitive
microelectrodes to determine the distribution of free
and bound calcium in the rabbit lens but, more
importantly, we develop techniques to investigate
active calcium extrusion from the lens.
Materials and Methods. Rabbit lenses (4-5 wk)
+60
+ 80
00
8
7
6
5
4
3
pCa
Fig. 1. Calibration curve for Ca++-sensitive microelectrode plots
the pCa values as a function of the potential difference (mV)
between the intracellular voltage and calcium-sensitive electrode
(filled circles). A typical value from one lens measurement (x) is
+45 mV, corresponding to a pCa of 4.5 (pCa = -logio [Ca++]) or
32 fiM.
containing elevated levels of calcium are obtained by
culturing lenses at 22°C in a HEPES-buffered medium
(pH = 7.4) containing 5 raM glucose, 135-150 mM
NaCl, 8 mM KC1, 3 mM MgCl2, and 20 mM CaCl 2 .
Temperature reduction has been shown to facilitate
a net accumulation of calcium without introducing
poisons or metabolic inhibitors.1 Moreover, changes
in the concentration of Na + and K + are minimized,
particularly as compared with temperature reductions
to 4°C. Osmolarity is adjusted to 300 ± 1 mOsm by
varying the NaCl content. Calcium extrusion experiments are performed in TCI99 at 37°C.
Calcium-sensitive microelectrodes are made according to methodology employed by Jacob, 4 and
suitable electrodes are in the resistance range of 80200 megohms. All electrodes, voltage and ion-sensitive, are calibrated before and after lens measurements
using a series of pCa buffers according to the method
of Maraben et al,6 who introduced the terminology
pCa to describe logarithmically the molar Ca ++ concentration. Thus the pCa3 buffer consists of 1 mM
CaCl 2, no ligand, 98 mM KC1, and 10 mM MOPS
(3-[N-morpholino]propanesulfonic acid, Sigma M1254) at pH 7.3. The pCa buffers 4, 5, 6, 7, and 8 all
contain 5 mM CaCl2, 90 mM KC1, 10 mM ligand
(NTA, NTA, HEDTA, EGTA, and EGTA, respectively) and 10 mM buffer—HEPES, pH 7.39; Tris,
pH 8.42; HEPES, pH 7.7; HEPES, pH 7.29; and
HEPES pH 7.8; respectively. The pCaoo solution
consists of 10 mM EGTA, 100 mM KC1, and 10
mM HEPES at pH 7.6. Following pH adjustment
with 1 N KOH, each buffer is passed through a 0.22ixm filter and stored in plastic at 4°C when not
in use.
In a typical experiment, immediately following
electrode calibration, the lens is impaled with the
reference microelectrode and the potential is monitored on channel A relative to an external reference
electrode in the media. Within 10 min of a stable
lens voltage, the calcium electrode is hydraulically
manipulated into place and this potential is simultaneously monitored on channel B. We can also monitor
the difference in potential (A - B) of the two electrodes
as the calcium potential. Figure 1 shows a standard
calibration curve (solid circles). The result of a typical
lens measurement is also illustrated (X) in which the
value of +45 mV is obtained from the differential
amplifier as the difference between the reference
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Reports
No. 7
Table 1. Intracellular Ca ++ in rabbit lens: influence of age and voltage*
Lens
condition
Culture
time
(hr)
[Ca++]r
Young (4 wk)
Mature (3 yr)
Traumatized^
Ouabain£
0
0
0
6
(nMtf
[CV + ] F
(nMtf
[Ca++]r
[Ca++]F
[Na+]T
(mM)\
250 ± 50
499 ± 50
250 ± 50
187 ± 18
33.3 ± 2.5
51.3 ±6.8
34.0 ± 3.0
29.9 ±4.1
7.58
9.73
7.35
6.25
13.0 ±0.05
21.8 ± 3.45
13.0 ±0.05
42.1 ± 1.86
potential of - 6 0 mV and the calcium-electrode potential of -105 mV. This is equivalent to pCa 4.5
(—log10 [Ca ++ ] = 4.5) and corresponds to a calcium
concentration of 32 /*M.
The concentration of total calcium and other ions
in the lens is measured by atomic absorption spectrophotometry (Perkin-Elmer, Model 272; Norwalk, CT)
as detailed elsewhere.7
The use of animals in the present study conforms
to the ARVO Resolution on the Use of Animals in
Research.
Results and Discussion. The free calcium concentration in the young rabbit lens of 33 /uM (Table 1)
obtained in the present study represents 13% of the
total and is significantly higher than 1 or 5 juM
obtained in rat and frog lenses, respectively.3'4 This
concentration is also approximately 100-fold greater
than that found in nerve and muscle and might be
expected to induce cytotoxic effects. For example, 10
nM levels close gap junctions in salivary gland epithelia.8 It should be remembered, however, that measurements from the lens were made in fiber cells, not
metabolically active epithelial cells which contain an
abundant supply of Ca-ATPase and are presumably
capable of extruding more calcium.
In view of the fact that this represents an unexpectedly high value for free calcium in the rabbit
lens, we carried out calcium measurements under
different conditions. Also, since the measurement of
calcium-sensitive voltages depends on the magnitude
of the membrane potential, we determined whether
free calcium measurements were influenced by
Recovery
condition^
0 time
6 hr
20 hr
-64.8
-64.0
-53.0
-50.0
± 0.84
±0.71
± 3.00
± 1.70
% Vitreous removed.
£ Cultured in 0.1 mM ouabain/TC199 at 37°C.
* Values are expressed as means ± 1 SE for n = 4-8 lenses.
t T signifies total; F signifies free, intracellular.
Table 2. Recovery of Ca
Voltage (m V)
changes in the potential. In addition, we measured
the voltages as a function of electrode depth. Table 1
shows that the free calcium measured in control and
traumatized lenses were identical despite a 12 mV
depolarization of membrane potential. In fact, a 25
mV depolarization induced by ouabain did not lead
to a significant difference in the measurement. It is
noteworthy that in the latter case the internal sodium
concentration had increased threefold following ouabain poisoning, but the total calcium had not changed
significantly.
When an electrode first penetrates the lens, whether
it is an intracellular or ion-sensitive electrode, the
reading is not stable until an electrode seal is obtained.
In the rabbit lens, steady values were obtained for
both electrodes after a depth of approximately 20 /u.m
was reached. In the present series of experiments, the
free calcium values were stable for more than 10 min
and did not in fact change significantly between
depths of 25 and 250 /um. This indicates that the
measurements were not contaminated by an extracellular leak component or influenced by tip blockage.
For convenience, an electrode depth of 100 /urn was
chosen for experimental purposes since very stable
values were obtained at this depth while the electrode
tended to block or break if depths greater than 300
/zm were attempted.
Previously, Hightower and Harrison9 have shown
that the total calcium content of the rabbit lens
increased progressively with age. Subsequent studies
on lens homogenates indicated that calcium binding
sites may have increased with age. The present study
-loaded lens*
[Ca++]T
[Ca++ h
(vM).
[Ca++]T
[Ca++]F
(mM)%
Voltagi?(mV)
232.5 ± 54
83.7 ± 16
54.0 ± 7.0
6.5
8.5
10.2
13.0 ±0.22
14.7 ±0.56
17.6 ± 1.06
-51.8 ± 1.4
-57.0 ±2.3
-55.0 ±2.0
t
1,505 ±0.07
714 ±0.06
555 ± 0.04
* Values are expressed as means ± 1 SE for n = 4-8 lenses.
t All lenses precultured for 24 hr in 20 mM Ca ++ , at 22°C; recovery in
TCI99 at 37°C.
% T signifies total; F signifies free, intracellular.
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / J u l y 1985
b^
a —
|'.I!::::::
I :::::£:t'iiiii!
'
iiiii
I .-ni z ----- -S.lllllliS :::::::::: ,1
=-.
fffffffi
irifi
| ::::::*
-
•
-
.
—
d ~
'Illir .tttti
1
:S:::::: II
tty^^iiiiiiiiitiiy
-
Fig. 2. Photographs of young rabbit lenses during incubation for
various periods of time in HEPES buffered media at 22°C containing
20 mM Ca ++ : a, zero time; b, 6 hr; c, 24 hr; d, 24 hr followed by
20 hr recovery in TC199 at 37°C.
shows in Table 1 that both free and total calcium
levels nearly double over a 3-year span, while the
ratio of total/free increases by a factor of 1.3. This
may suggest that redistribution of calcium occurs in
the mature lens. It is interesting that this increase in
internal calcium is not accompanied by a change in
membrane potential as is typically characteristic of a
change in membrane permeability.
The level of free calcium in the lens represents a
balance between the movement of Ca 2+ across the
surface membranes and the exchange between internal
binding sites. Calcium movements across the lens
membranes have hitherto been studied exclusively
using 4SCa radioisotope methods, and recently, Paterson and Delamere5 have shown that such studies are
extremely difficult to interpret in view of the slow
diffusion of calcium into the lens. To circumvent the
shortcomings of tracer techniques in the study of
active transport, we have used the ion-sensitive calcium electrode to monitor the movement of intracellular free calcium from the lens against an electrochemical gradient.
Lenses were first precultured in a calcium-enriched,
Hepes-buffered medium for 20 hr. During this time,
the total calcium in the lens increased from 250 to
1,505 fxM, while the free calcium increased from 33
to 232 yM (Table 2). During this incubation, there
Vol. 26
was little change in sodium content and only a 10
mV depolarization in membrane potential. There
was no significant hydration of the lens as in previous
experiments, but the lens cortex was opaque at the
end of the incubation period (Fig. 2). During subsequent culture in TCI99 at 37 ° Q the voltage hyperpolarized to within 85% of the control value (Table
2), and lens transparency was restored. More importantly, there occurred a decrease in free calcium from
232 to 84 ^M within 6 hr and a further decline to
54 uM at 20 hr. Intracellular calcium was therefore
extruded from the lens against a significant electrochemical gradient, indeed, under conditions where
the electrochemical gradient was increasing. Since the
external calcium concentration is 2,000 ;uM and the
membrane potential was nearly —60 mV calcium
extrusion occurred against a thermodynamically
equivalent concentration ratio of 9,000. This is unequivocal evidence that active calcium extrusion takes
place in the rabbit lens.
Key words: intracellular calcium, transport, microelectrode,
lens, opacification
From the Institute of Biological Sciences,* Oakland University,
Rochester, Michigan; and the School of Biological Sciences,!
University of East Anglia, Norwich, United Kingdom. Supported
by National Eye Institute research grant EY-03680. Submitted for
publication: October 19, 1984. Reprint requests: Kenneth R. Hightower, PhD, Institute of Biological Sciences, Oakland University,
Rochester, Ml 48063.
References
1. Hightower KR and Dering M: Development and reversal of
lens opacification caused by calcium. Invest Ophthalmol Vis
Sci 25:1108, 1984.
2. Hightower KR: The influence of calcium on protein synthesis
in the rabbit lens. Invest Ophthalmol Vis Sci 24:1422, 1983.
3. Duncan G and Jacob TJC: Calcium and the physiology of
cataract. 1984 Human Cataract Formation. Pitman, London,
Ciba Foundation Symposium 106, in press.
4. Jacob TJC: A direct measurement of intracellular free calcium
within the iens. Exp Eye Res 36:451, 1983.
5. Paterson CA and Delamere NA: An analysis of 45Ca fluxes in
the rabbit lens. Curr Eye Res 2:727, 1982/1983.
6. Maraben E, Rink TJ, Tsien RW, and Tsien RY: Free calcium
in heart muscle at rest and during contraction measured with
Ca++-sensitive microelectrodes. Nature 286:845, 1980.
7. Hightower KR, Leverenz V, and Reddy VN: Calcium transport
in the lens. Invest Ophthalmol Vis Sci 19:1059, 1980.
8. Lowenstein WR and Rose B: Calcium in (junctional) intercellular communication and a thought on its behavior in intracellular communication. Ann NY Acad Sci 307:285, 1978.
9. Hightower KR and Harrison SE: Changes in the Ca ++ , Mg ++
and SH content in the developing rabbit lens. Lens Res 1:249,
1983.
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