Yonago Acta medica 1999;42:51–59

Effects of Extracellular Acidification on Intracellular pH and ATP-Induced
Calcium Mobilization in Rabbit Lens Epithelial Cells
Akiko Narita*†, Kenji Yawata*†, Masao Nagata†, Yasuaki Aoyama* andYasuaki Kawai**Second Department of Physiology and †Department of Ophthalmology, Faculty of Medicine,
Tottori University, Yonago 683-0826, Japan
Effects of extracellular acidification on intracellular pH (pHi) and ATP-induced calcium
mobilization were investigated in rabbit lens epithelial cells. Primary-cultured lensepithelial cells of Japanese white rabbits were used. Intracellular calcium ([Ca2+]i) andpHi were measured by using fluorescent dyes, fura-2 acetoxymethylester (fura-2 AM)
and 2',7'-bis (carboxyethyl)-5,6-carboxyfluorescein acetoxymethylester (BCECF AM),
respectively. The addition of 10 µmol/L ATP produced an initial peak followed by asustained increase in [Ca2+]i in a standard artificial aqueous humor at extracellular pH(pHo) 7.40. The initial peak was abolished by pretreatment with 1 µmol/L thapsigargin,
whereas the sustained increase was attenuated in a Ca2+-free solution or by pretreatment
with 100 µmol/L verapamil. Acidification of the pHo from 7.40 to 6.80 decreased thepHi from 7.21 to 7.03, and enhanced both the initial peak and sustained increase in[Ca2+]i. These results suggest that acidification of pHo significantly affects the pHi andmodifies the ATP-induced [Ca2+]i transient in rabbit lens epithelial cells.
Key words: ATP-induced calcium mobilization; extracellular acidification; intracellular pH; rabbit
lens epithelial cells

Concentration of cytosolic free calcium ([Ca2+]i)
modulates a variety of cell functions such asmuscle contraction, secretion, phototransduction and cell proliferation. A large number ofagonists are known to regulate the [Ca2+]i by
activating phosphatidylinositol turnover(Berridge, 1993). They include acetylcholine,
histamine, adrenaline, arginine vasopressin,
bradykinin and ATP. ATP has been reported to
be present in the normal aqueous humor and to
be released into it from injured cells (Neary etal., 1996). It is also known that ATP exhibits its
action through P2u purinergic receptor in humanlens epithelial cells (Riach et al., 1995).
Intracellular pH (pHi) also plays an important role in the regulation of cellular functions.
Previous studies demonstrated that [Ca2+]i andpHi are linked to each other, although the direction of changes in those parameters is controversial. A parallel relationship was observed
between [Ca2+]i and pHi in squid axons (Baker,1978), rat lymphocytes (Grinstein and Goetz,1985), cultured vascular smooth muscles (Siskind
et al., 1989) and colonic carcinoma cell line
HT29 (Benning et al., 1996). On the other
hand, an inverse relationship between them wasfound in Xenopus embryos (Rink et al,. 1980),
sheep heart Purkinje fibers (Bers and Ellis,
1982), rat vascular smooth muscles (Daugirdas
et al., 1995) and rat parotid acinar cells(Nishiguchi et al., 1997). In the rat lens, however, the acidification of pHi had no effect on[Ca2+]i (Bassnett and Duncan, 1988). The effectof pHi on agonist-induced [Ca 2+]i transients

Abbreviations: BCECF AM, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein acetoxymethylester; [Ca2+]i,
concentration of cytosolic free calcium; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine
serum; fura-2 AM, fura-2 acetoxymethylester; IP3, inositol triphosphate; pH i , intracellular pH; pH o,extracellular pH

51

A. Narita et al.

cell suspension (0.5 mL) was placed into a 35mm culture dish containing a 10 mm diameterglass coverslip coated with poly-d-lysine(MatTek Corp., Ashland, MA) and allowed tosettle. The cells were flooded with DMEM
containing 10% FBS, which was changed twicea week, and cultured for 10 to 21 days during
which confluent cultures were usually obtained.

was also investigated in HT29 cells (Nitschke etal., 1997) and rat lacrimal acinar cells (Yodozawa
et al., 1997), but not in lens epithelial cells.
Therefore, in the present study effects ofextracellular pH (pHo) on pHi and ATP-induced
calcium mobilization were investigated in
rabbit lens epithelial cells by measuring [Ca2+]iand pHi simultaneously with fluorescent dyes,
fura-2 acetoxymethylester (fura-2 AM) and 2',7'-bis (carboxyethyl)-5, 6-carboxyfluorescein
acetoxymethylester (BCECF AM), respectively. The main questions were: i) Are the ATPinduced [Ca2+]i transients modified by an acidification of pHi? and ii) Which calcium source
is involved in such pH-dependent alteration?

Dye loading and fluorescence measurement
Cells were loaded with the fluorescent dyes 15µmol/L fura-2 AM (Dojindo Laboratories,
Kumamoto, Japan) and 1.5 µmol/L BCECF
AM (Dojindo Laboratories) in artificial aqueous humor at 37°C in the dark for 1 h. The composition of standard artificial aqueous humor in
mmol/L was as follows: 124 NaCl, 5 KCl, 1
CaCl2, 0.5 MgCl2, 5 glucose and 10 HEPES.
The cells were washed 3 times and continuously perfused at a rate of 2 mL/min for morethan 40 min with artificial aqueous humor. The
culture dish was mounted on the heated stage ofan inverted epifluorescence microscope (TMD300; Nikon Corp., Tokyo) equipped with afluorometric system (QuantiCell 700, Applied
Imaging, Sunderland, United Kingdom). Cells
were observed through the coverslip of theculture dish using a 40 × 0.85 numericalaperture, dry objective lens (Fluor 40, Nikon
Corp.). Fura-2 AM and BCECF AM were
excited with light from a 100 W xenon lamp
which was alternatively filtered to 340 or 380
nm for fura-2 AM and to 440 or 490 nm for
BCECF AM excitation, respectively. The
fluorescence emission was filtered between 510
nm and 535 nm, and monitored with an intensified CCD camera.
The images were analyzed with the software
package Graphical User Interface (Applied
Imaging) which performed a backgroundsubtraction. Geometric regions matching
individual cells were defined and analyzed forchanges in fluorescence ratio. Concentration ofcytosolic free calcium was calculated from theratio (R) of the fluorescence measured withexcitation at 340 nm and 380 nm using thefollowing equation by Grynkiewicz and co-Materials and Methods

Animals
Japanese white rabbits weighing 2.5 to 3.5 kg
were used in this study. The eyeballs were
removed within 1 h after the animals were
killed by an injection of an overdose of sodium
pentobarbital (100 mg/kg, intravenously). Allprocedures were reviewed and approved by theCommittee for Animal Experimentation in the
Faculty of Medicine, Tottori University andadhered to the ARVO Statement for the Use of
Animals in Ophthalmic Vision Research.

Cell culture
The eyeballs were maintained in 50 mL ofphosphate-buffered solution at 5°C without
added Ca2+ and Mg2+. The lenses were dissected out and placed in 3 mL of Dulbecco’s modified Eagle’s medium (DMEM; Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) containing
10% fetal bovine serum (FBS) 1 to 3 h later.
The capsules with the epithelium were removed
and digested with 0.25% trypsin (Sigma
Chemical Co., St. Louis, MO) for 3 min at 37°C
in humidified atmosphere containing 5%
carbon dioxide. After adding 3.5 mL of DMEM
with 10% FBS to slow the digestion, themedium with the capsule was triturated 4 to 5
times to disperse cells from the capsules. The
52

pHi and ATP-induced calcium mobilization[Ca2+]i[Ca2+]i[Ca2+]iworkers (1985): [Ca2+]i = Kd × (R – Rmin)/(Rmax
Solution and agonist application– R) × β, where Kd is the dissociation constantof the fura-2 AM; Rmax and Rmin are the ratios During the experiment cells were covered withfor the bound and unbound forms of the fura-2 3 mL artificial aqueous humor and perfused
AM/Ca2+ complex; and β is the ratio between with the same solution using a peristaltic pump
the maximum and the minimum fluorescence (EYELA MICROTUBE PUMP MP-3, Tokyo
intensities of fura-2 AM at 380 nm excitation. Rikakikai Co., Ltd., Tokyo) at a flow rate of 2
To obtain R max, the cells were exposed to a mL/min. The pH of the solution was adjusted atsolution of the following composition: 150 either 7.40 or 6.80 by adding 1 N NaOH. The
mmol/L KCl, 10 µmol/L ionomycin (Sigma Ca2+-free solution was made by substituting 2
Chemical), 10 mmol/L HEPES and 10 mmol/L mmol/L EGTA for 1 mmol/L CaCl 2 in the
CaCl2. The cells were then exposed to the Ca- standard artificial aqueous humor. Each agonfree solution with 1 mmol/L EGTA to obtain ist was dissolved just before use and added toRmin. The value for the Kd increases signifi- the perfusate. The doses of agonists were excantly when pH falls to less than 6.50. How- pressed as the final organ bath concentrations.
ever, acidification from 7.20 to 7.0 has amuch smaller effect on the fura-2 AM(nmol/L)
Kd value (Negulescu and Machen, 1990; A 2500
100 µmol/L ATP
Battle et al., 1993) causing less than 10%
2000
underestimation of [Ca 2+ ] i. In the1500
present study, the constant value (2241000
nmol/L) determined by Grynkiewicz
500
and coworkers (1985) was used since
pHi did not fall below 7.0.
0
0
50
100
150
200
250 (s)Intracellular pH was estimated as aTime
490/440 ratio of the fluorescence andcalibrated as follows: at the end of each B 2000(nmol/L)
100 µmol/L acetylcholine
experiment the cells were exposed to 10
1500µmol/L nigericin, which equilibrates thepHi with the known pHo (Williums et al.,10001992), dissolved in a potassium (150500
mmol/L) buffer. The 490/440 ratios
were obtained during changes in pHo by0
perfusing three pH standard solutions. A
0
50
100
150
200
250 (s)pH standard (pH i 6.50) contained 10
Time
mmol/L piperazine-N,N,-bis(2-etaeth(nmol/L)
sulfonic acid) (PIPES, Sigma Chemical C 2000
1 µmol/L bradykininCo.), and other standards (pHo 7.0 and
15007.50) contained 10 mmol/L HEPES. As
1000
the response ratio was linear in the pHo
range between 7.50 and 6.50, a simple
500
transformation was performed to obtainthe corresponding pHi values from the
0
0
50
100
150
200
250 (s)ratios (Williums et al., 1992). All the exTime
perimental protocols gave pHi values
2+
within the linear range. In order to mini- Fig. 1. Typical responses of [Ca ] i in primary-cultured
mize the bleaching effect, excitation of rabbit lens epithelial cells during continuous perfusion with
100 µmol/L ATP (A), 100 µmol/L acetylcholine (B) or 1
BCECF AM was not carried out in some µmol/L bradykinin (C). [Ca2+] , concentration of cytosoliciexperiments.
free calcium.
53

A. Narita et al.

Table 1. Effects of various agonists on [Ca2+]i in primary-cultured rabbit lens epithelial cells(n = 6) at pHo 7.40
AgonistConcentration(µmol/L)

Peak ∆[Ca2+]i(nmol/L)†

Responded cells(%)*

Acetylcholine
10087866 ± 264
ATP
100
100
1227 ± 218
Bradykinin
183475 ± 178
Histamine
100
20
68 ± 28
Dopamine
10000Adrenaline
10000Angiotensin II100* Percentage of cells which responded to each agonist.
† Values are expressed as mean ± SEM.
[Ca2+]i, concentration of cytosolic free calcium; peak ∆[Ca2+]i, peak increases in [Ca2+]i; pHo, extracellularpH.

During the application of the agonist the perfusion rate was increased to 5 mL/min to achievea quick delivery of the agonist. Changing theflow rate itself had no effect on [Ca2+]i. In order
to avoid desensitization, consecutive agonist
applications were made with intervals of longer
than 30 min each. The following agonists wereused in this study: acetylcholine, ATP, bradykinin, histamine, dopamine, adrenaline andangiotensin II, which were all purchased from
Sigma Chemical Co. When the cells were treated with verapamil or thapsigargin (also purchased from Sigma Chemical Co.), the antagonist was added in the perfusate 15 min before
the application of ATP.

Statistical analysis
The values are expressed as mean ± SEM. The
data of [Ca 2+ ] i and pH i were obtained byaveraging the signals from 5 single cells in each
culture dish. The n values reflect the number ofanimals used. Student’s t-test (unpaired) wasapplied to determine the statistical difference
between the 2 groups. Values of P < 0.05 wereconsidered to be statistically significant.

Results
The resting [Ca2+]i was 133 ± 10 nmol/L (n =12) in primary-cultured rabbit lens epithelial
cells perfused with the standard artificial
aqueous humor. The pHi was 7.21 ± 0.01 (n =5) when pHo was 7.40.
Figure 1 shows typical responses of [Ca2+]iin the rabbit lens epithelial cells during continuous perfusion with 100 µmol/L ATP (Fig.
1A), 100 µmol/L acetylcholine (Fig. 1B) or 1
µmol/L bradykinin (Fig. 1C) in the standard
artificial aqueous humor. Administration of
ATP or acetylcholine produced an initial peakfollowed by a sustained increase in [Ca 2+]i,
whereas bradykinin caused only the initial
peak. The peak increases in [Ca2+]i (∆[Ca2+]i)
produced by each agonist are shown in Table 1.
Adrenaline, dopamine and angiotensin II had
little effect on [Ca2+]i in the rabbit lens epithe-(nmol/L)∆[Ca2+]i

1500

1000

500

0
0.1

1

10

100

(µmol/L)ATP

Fig. 2. Concentration-response relationship of ATPinduced ∆ [Ca2+] i in primary-cultured rabbit lensepithelial cells (n = 6). ∆[Ca2+]i, increases in [Ca2+]i.

54

pHi and ATP-induced calcium mobilizationA(nmol/L)

10 µmol/L ATP

2000∆[Ca2+]i

pHo 7.40
pHo 6.80

1500

1000

500

0
0

B

50

100

150

200

250

(s)

150

200

250

(s)

Time
7.8

7.6

pHi

7.4

7.2

7.0

6.8

6.6
0

50

100

Time

Fig. 3. Effects of extracellular acidification on 10 µmol/L ATP-induced [Ca2+]i transient (A) and intracellular
pH (pHi) (B). [Ca2+]i, concentration of cytosolic free calcium; pHo, extracellular pH.

peak and 760 ± 95 nmol/L at the sustainedphase (4 min after the administration of ATP)when pHo was 6.80, which were significantly
greater than the values at pHo 7.40 (peak: 1090
± 173 nmol/L, P < 0.05; sustained phase: 423 ±67 nmol/L, P < 0.05) (Fig. 4).
The source of calcium mobilization induced
by ATP was investigated in the rabbit lens epithelial cells. The addition of 10 µmol/L ATP
produced an initial peak followed by a sustained increase in [Ca2+]i in the standard artificial
aqueous humor (Fig. 5A). In the Ca2+-freesolution, 10 µmol/L ATP produced the initial
increase in [Ca 2+]i without the sustained increase (Fig. 5B). The sustained increase induced by 10 µmol/L ATP was also greatly attenuated after the cells had been pretreated with 100µmol/L verapamil, a L-type calcium channellial cells. Histamine caused a small increase of[Ca2+]i in limited number of the cells.
Figure 2 shows the concentration-response
relationship of ATP-induced ∆[Ca2+]i in the rabbit lens epithelial cells. The threshold concentration for this response was 0.1 µmol/L. The
EC50 value, the concentration of the agonist
causing half of the maximum response, was3.25 ± 0.58 µmol/L. Figure 3 demonstrates theinfluence of extracellular acidification on 10µmol/L ATP-induced changes in [Ca2+]i and
pHi. Changing extracellular pH from 7.40 to
6.80 decreased pHi by 0.18 units (Fig. 3B), butdoes not alter the resting [Ca2+]i (Fig. 3A). This
acidification enhanced the magnitudes of boththe initial peak and the sustained increase in[Ca2+]i induced by ATP. ATP (10 µmol/L) increased [Ca2+]i by 1532 ± 101 nmol/L at the55

A. Narita et al.

(nmol/L)(nmol/L)∆[Ca2+]i

2000

2000

P < 0.05

1500

1500

1000

1000

500

500

Discussion
In a previous study (Duncan et al., 1996)[Ca 2+]i in rabbit lens cells was measured byaveraging the signals from a large number ofcells using a cuvette-based fluorimeter systemsince it was not possible to incorporate sufficient fura-2 AM into the cells to image single
cells. In the present study, therefore, we loaded
the cells with a high concentration (15 µmol/L)of fura-2 AM for a long period (1 h). Thus, theconcentration is higher than that usually used
(1–5 µmol/L), and the period is longer thanusual (20–45 min) in lens cells (Riach et al.,
1995; Duncan et al., 1996; Churchill and Louis,
1997).
The present results showed that ATP,acetylcholine and bradykinin caused a marked
increase of [Ca2+]i in the cultured rabbit lensepithelial cells, whereas histamine, dopamine,
adrenaline and angiotensin II had little or noeffect on the [Ca2+]i (Table 1). Duncan andcoworkers (1996) demonstrated that histamine
but not acetylcholine produced a marked
elevation of [Ca2+]i in a rabbit lens cell line
(NN1003A), which is opposite to our results.

P < 0.05

00
pH 6.80

pH 7.40

pH 6.80

Peak

pH 7.40

Sustained increase

Fig. 4. Effects of extracellular acidification on 10µmol/L ATP-induced peak and sustained increasesof [Ca2+]i in primary-cultured rabbit lens epithelial
cells (n = 6). [Ca2+]i, concentration of cytosolic freecalcium; ∆[Ca2+]i, increases in [Ca2+]i.

blocker (Fig. 5C). Application of 1 µmol/Lthapsigargin, a Ca2+ pump inhibitor, induced agradual and sustained elevation of [Ca 2+]i to
874 ± 31 nmol/L (n = 6) (Fig. 5D). In thethapsigargin-treated cells, 10 µmol/L ATP
failed to produce both the initial peak and thesustained increase in [Ca2+]i (Fig. 5D).

(nmol/L)
2000

B

10 µmol/L ATP

1500[Ca2+]i[Ca2+]i

A

1000

1000

0

0
0
(nmol/L)
2000

100

200

Time

300 (s)

0

100

200

300

400

500 (s)Time

D

100 µmol/L verapamil(nmol/L)
2000

10 µmol/L ATP
1500[Ca2+]i[Ca2+]i

10 µmol/L ATP

1500

500

500

C

Ca2+-free(nmol/L)
2000

1000

500

1 µmol/L thapsigargin
10 µmol/L ATP

1500

1000

500

0
0

500

1000

1500

(s)

0
–1000

–500

0

500 (s)

Time

Time

Fig. 5. Responses of [Ca2+]i in primary-cultured rabbit lens epithelial cells to 10 µmol/L ATP in a standard
artificial aqueous humor (A), in a Ca2+-free solution (B) or by treatment with 100 µmol/L verapamil (C) orwith 1 µmol/L thapsigargin (D). [Ca2+]i, concentration of cytosolic free calcium.

56

pHi and ATP-induced calcium mobilizationin rat lens (Bassnett and Duncan, 1988) or incanine tracheal smooth muscle (Yamakage etal., 1995) but is comparable to that in rat portal
vein (Taggrart et al., 1994) or guinea-pig vas
deferens (Aickin, 1984). Changes in pHi may
be buffered by various proteins and phosphates.
In addition, Na+-H+ and Cl–-HCO3– exchangesystems may provide further pHi regulation inlens epithelial cells (Williums et al., 1992). The
small decrease of pHi observed in the present
study may imply that such buffer and exchangesystems are well preserved.
The acidification of pH i significantly enhanced both the initial peak and the sustained
increase induced by 10 µmol/L ATP in rabbit
lens epithelial cells (Fig. 4). This is consistent
with the result obtained in HT29 cells that an
intracellular acidification enhanced the peak
and plateau [Ca2+]i transients elicited by carbachol (Nitschke et al., 1997). The initial peak in
ATP-induced [Ca2+]i transient is probably due
to Ca2+ release from intracellular stores activated by a second messenger, inositol triphosphate (IP3). It has been shown that rabbit
lens epithelial cells have a functional phosphoinositide cycle (Vivekanandan and Lou, 1989).
The action site of pHi seems to be distal to the
IP 3 production because the IP 3 production
caused by carbachol was unaltered by theacidification of pHi (Nitschke et al., 1997).
Intracellular acidification also seems to modify
the Ca2+ influx through the L-type Ca2+ channel
since the sustained increase in [Ca2+]i caused by
ATP was enhanced by the acidification. The
similar finding, that cytosolic acidification
stimulates an influx of Ca2+, was observed inChlamydomonas (Quarmby, 1996)
The relationship between calcium and cataract has been discussed for a long time (Duncan
and Jacob, 1994). It is generally accepted thatan increase in lens calcium plays a certain rolein the development of cataract. An elevation of[Ca 2+]i seems to change membrane permeability (Bernardini and Perrachia, 1981) andstability of the lens cytoplasmic gel (Duncan
and Jacob, 1994). Thus, clarifying the mechanism for [Ca2+]i regulation may provide clinically important information as well as advances in physiological knowledge.

The reason for this discrepancy is not clear.
Changes in type and/or number of receptors,
which may occur as a result of adaptation in
culture medium, may explain the difference.
Both studies, on the other hands, revealed that
ATP induced a marked increase of [Ca2+]i in
rabbit lens cells.
The addition of ATP ranging from 0.1 to100 µmol/L caused a concentration-dependent
increase in [Ca2+]i (Fig.2). When the effect ofpH on the ATP-induced [Ca2+]i transient was
examined, 10 µmol/L of ATP, a concentration
which caused a submaximum response (Fig. 2),
was used. Continuous perfusion with 10 µmol/
L ATP produced an initial peak followed by asustained increase in [Ca 2+]i (Fig.5A). The
initial peak was abolished by pretreatment with
1 µmol/L thapsigargin but was not affected in aCa2+-free solution or by pretreatment with 100µmol/L verapamil, indicating that release of
Ca2+ from intracellular stores was involved. On
the other hand, the influx of extracellular
calcium through the L-type Ca 2+ channel isprobably responsible for the sustained increase
in [Ca2+]i because it was abolished in the Ca2+free solution and significantly attenuated by
pretreatment with verapamil. Duncan and coworkers (1996) reported that 1 µmol/L ATP
produced only a transient increase in [Ca2+]i in
rabbit lens cells. The failure for ATP to produce a sustained increase may be explained by
the differences in concentration and applicationof ATP. A lower concentration (1 µmol/L) of
ATP was added by a single injection in theirexperiments, whereas a higher concentration(10 µmol/L) of ATP was continuously applied
over several minutes in the present study. Bothtransient and sustained increases in [Ca2+]i wereelicited in human (Riach et al., 1995) and sheep(Churchill and Louis, 1997) lens epithelial cells
which were continuously exposed to highconcentrations (10–100 µmol/L) of ATP.
The pHi value measured in the present study
was 7.21 ± 0.01 when the pHo was maintained
at 7.40. This is consistent with the previous
reports using human (Sophie et al., 1988) andbovine (Williums et al., 1992) lens cells. Lowering the pHo to 6.80 decreased the pHi to 7.03.
This change in pHi is smaller than that observed57

A. Narita et al.

10 Daugirdas JT, Arrieta J, Ye M, Flores G, Battle
DC. Intracellular acidification associated withchanges in free cytosolic calcium: evidence forCa2+/H+ exchange via a plasma membrane Ca2+ATPase in vascular smooth muscle cells. J Clin
Invest 1995;95:1480–1489.
11 Duncan G, Jacob TJC. Calcium, cell signallingand cataract. Prog Retinal Eye Res 1994;13:623–652.
12 Duncan G, Riach RA, Williums MR, Webb SF,
Dawson AP, Reddan JR. Calcium mobilization
modulates growth of lens cells. Cell Calcium
1996;19:83–89.
13 Grinstein S, Goetz JD. Control of free cytoplasmic calcium by intracellular pH in rat lymphocytes. Biochim Biophys Acta 1985;819:267–
270.
14 Grynkiewicz G, Poenie M, Tsien RY. A newgeneration of Ca 2+ indicators with greatly improved fluorescent properties. J Biol Chem 1985;
260:3440–3450.
15 Neary JT, Rathbone MP, Cattabeni F, Abbracchio
MP, Burnstock G. Trophic actions of extracellular nucleotides and nucleosides on glial andneuronal cells. Trends Neurosci 1996;19:13–18.
16 Negulescu PA, Machen TE. Lowering extracellular sodium or pH raises intracellular calcium
in gastric cells. J Membrane Biol 1990;116:239–
248.
17 Nishiguchi H, Hayashi T, Shigetomi T, Ueda M,
Tomita T. Changes in intracellular Ca2+ concentration produced by the alteration of intracellular
pH in rat parotid acinar cells. Jpn J Physiol 1997;
47:41–49.
18 Nitschke R, Benning N, Ricken S, Leipziger J,
Fischer KG, Greger R. Effect of intracellular pH
on agonist-induced [Ca 2+]i transients in HT 29
cells. Pflügers Arch-Eur J Physiol 1997;434:
466–474.
19 Quarmby LM. Ca2+ influx activated by low pH
in Chlamydomonas. J Gen Physiol 1996;108:351–361.
20 Riach RA, Duncan G, Williums MR, Webb SF.
Histamine and ATP mobilize calcium by activation of H1 and P2u receptors in human lens epithelial cells. J Physiol 1995;486:273–282.
21 Rink TJ, Tsien RY, Warner AE. Free calcium in
Xenopus embryos measured with ion-selective
microelectrodes. Nature 1980;283:658–660.
22 Siskind MS, McCoy CE, Chobanian A, Schwartz
JH. Regulation of intracellular calcium by cell
pH in vascular smooth muscle cells. Am J Physiol 1989;256:C234–C240.
23 Sophie S, Duncan G, Julia MM, Alan RP. Membrane and communication properties of tissue
cultured human lens epithelial cells. Invest Ophthalmol Vis Sci 1988;29:1713–1725.
24 Taggrart M, Austin C, Wray S. A comparison of
the effects of intracellular and extracellular pH on

The present results demonstrated that ATP
produced an initial peak followed by a sustained increase of [Ca2+]i in the cultured rabbit lensepithelial cells, and that extracellular acidification reduced pHi and enhanced both the peakand sustained [Ca2+]i transients. These results
suggest that pHo acidification reduces pHi andmay modify intracellular signal transduction
and membrane property which in turn affectcellular function in lens cells.

Acknowledgements: We thank Prof. Akihiko Tamai,
Dept. of Ophthalmology, Faculty of Medicine,
Tottori University, for his critical reading of themanuscript.

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(Received December 18, Accepted December 28, 1998)

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