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Cytoplasmic pH in the action of epidermal growth factor (EGF) in cultured porcine thyroid cells
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 157, No. 1, 1988
Pages 346-349
November 30,1988
CYTOPLASMIC pH IN THE ACTION OF EPIDERMAL GROWTH FACTOR (EGF) IN CULTURED PORCINE THYROID CELLS Nobuyuki
Takasu,
Yoshitaka
Nagasawa, Ichiro Yoshifusa Shimizu
Komiya,
Takashi
Yamada and
Department of Gerontology, Endocrinology and Metabolism, School of Medicine, Shinshu University, Asahi 3 - I - I , Matsumoto, Nagano-ken 390, Japan Received
October
20,
1988
We demonstrate measurement of cytoplasmic pH (pHi), using 2 ' , 7 ' - b i s ( 2 carboxyethyl)-5 (and 6-) carboxyfluorescein (BCECF), an internalized fluorescent pHi i n d i c a t o r , in thyroid c e l l s . Using cultured porcine thyr9id cells, we studied the e f f e c t s of epidermal growth f a c t o r (EGF) on pHi and [~H] thymidine incorporation; lO nM EGF a l k a l i n i z e s thyroiQ c@lls and stimulates thymidine incorporation. The r e s u l t s indicate that Na~/HT exchange or c e l l a l k a l i n i z a t i o n may function as a transmembrane signal transducer in the action of EGF in the thyroid c e l l s . ® 1988 Academic P. . . . . I ....
The
mechanisms
proliferation rapidly cells,
by
are
activate
largely
growth factors unknown.
stimulate
Recent evidence suggests
cytoplasmic
pH (pHi) as a p o t e n t i a l
Recently, 2 ' , 7 ' - b i s ( 2 - c a r b o x y e t h y l ) - 5
(BCECF)
(6),
examine
the pHi-regulating mechanisms.
an
proliferation
alkalinization
cell
mitogens
alkalinize
"messenger" for
the cell
has
b e e n used to
Epidermal growth f a c t o r (EGF) is known (7-9).
is unknown. Using cultured porcine thyroid c e l l s , on pHi and thymidine incorporation.
that
and
(and 6-) carboxyfluorescein
i n t e r n a l i z e d fluorescent pHi i n d i c a t o r ,
to stimulate thyroid c e l l
cell
metabolism
Na+/H+ exchange in the plasma membrane and
implicating
growth ( I - 5 ) .
which
However,
the mechanism of t h i s
we studied the e f f e c t s of EGF
We intended to show that Na+/H+ exchange or
might function as a transmembrane signal
transducer
for
thyroid c e l l growth. MATERIALS AND METHODS ! h l r o i d cell c u l t u r e : Thyroid c e l l s were obtained from porcine thyroid gland~ as described previously (lO). Freshly isolated c e l l s were suspended (3xlO v cells/ml) in Eagle's minimum essential medium (MEM) supplemented with 0.5% 0006-291X/88
$1.50
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
346
VoI. 157, No. 1, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
f e t a l - c a l f serum and a n t i b i o t i c s ( p e n i c i l l i n , 200 units/ml; streptomycin, 50 ug/ml). Cells were cultured as a suspension at 370C in a 95% a i r : 5% CO2 water-saturated atmosphere ( I I ) , C~toplasmic ~H ~ H ~ : Cytoplasmic pH (pHi) was measured with the tetraacetoxymethyl ester (BCECF-AM) of 2 ' , 7 ' - b i s ( 2 - c a r b o x y e t h y l ) - 5 (and 6-) carboxyfluorescein (BCECF) (6). PoTcine thyroid c e l l s , cultured f o r 16 h, were washed twice in a standard HEPES-Na~ solution containing 140 mM NaCl, l mM KCI, l mM MgCl~, 1 mM CaCl2, lO mM glucose, and buffered with 20 mM HEPES, pH 7.2. The c e l l { were resuspended in the same b u f f e r containing 3 ~M BCECF-AM and incubated f o r 30 min at 37°C. BCECF-AMwas converted to the impermeant BCECF by cytoplasmic esterases (6). After loading, the c e l l s were washed twice i~ HEPES-Na" solution to remove e x t r a c e l l u l a r dye and were resuspended at 3xlO ~ cells/ml. The BCECF-loaded c e l l s were put into a thermostatic cuvette in a Hitachi F-4000 fluorometer (Hitachi, Tokyo), and the c e l l suspension was continuously s t i r r e d with a magnetic microbar. The pHi-dependent emission i n t e n s i t y was continuously recorded ( e x c i t a t i o n wavelength 500 nm; emission wavelength 530 nm; s l i t s 5.5/ll-nm bandpass). Calibration of cytoplasmic dye fluorescence as a function of pHl was obtained from H e a u ] l l b r a t l o n methods using the K+/H+ lonophore . . . .n l g e r l c l n , which sets [H÷ ] i / [ H + ]o equal to [ K + ] i / [ K + ] o as previously described (12). B r i e f l y , c a l i b r a t i o n was obtained by incubation of BCECF-loaded c e l l s in K~ medium (140 mM KCl, l mM NaCl, l mM MgClg, l mM CaCl~, lO mM glucose) buffered with 20 mM HEPES, and containing nige~icin ( 0 . 7 ~ g ~ l ) . The pHi-dependent emission i n t e n s i t y was then measured. IncorEoration of [~H~ th#midin~: Isolated thyroid c e l l s were cultured f o r 16 h and--then EGF and O : l ~ C i - o f - [ H] thymidine were added to measure the e f f e c t of EGF on thymidine incorporation. After incubation, the c e l l s were collected and washed three times with lOZ t r i c h l o r o a c e t i c acid (TCA) and dissolved in 0.3 N NaOH. T h e n t h e i r r a d i o a c t i v i t i e s were measured in a l i q u i d scintillation counter. Materials e t c . : Murine epidermal growth f a c t o r (EGF) was obtained from Collaborative Research (Waltman, MA, U.S.A.); n i g e r i c i n and HEPES from Sigma (St. Louis, MO, U.S.A.); fetal c a l f s~rum and Eagle's MEM from Flow Laboratories ( I r v i n e , Scotland, U.K.); [~H] thymidine (2 Ci/mmol) from New England Nuclear Corp. (Boston, MA, U.S.A.); BCECF-AMfrom Molecular Probes, Inc. (Junction City, OR, U.S.A.). BCECF-AM was dissolved in dimethyl s u l f o x i d e and stored at -20°C. All other chemicals were of the highest p u r i t y available commercially. Experiments were conducted at least 4 times. Typical data and the final concentration of EGF are shown in the t e x t and the f i g u r e . Data were s t a t i s t i c a l l y analyzed with analysis of variance (ANOVA). P
-
-
t
.
-
•
RESULTS EGF-stimulated thyroid c e l l a l k a l i n i z a t i o n and ~ m ~
~Q£~[~9 r a t i ° n ,
The c e l l s were loaded with BCECF and then pHi was recorded continuously (Fig. I).
The resting pHi of porcine thyroid c e l l s in HEPES-Na+ solution at 37°C was
7.22+0.04
(n=8).
alkalinization,
Addition
of lO nM EGF to these c e l l s induced
intracellular
reaching a pH of 7.39+0.06 (n=8) in 3 min.
Ten nM EGF produced a gradual increase in [3H] thymidine incorporation 2). A s i g n i f i c a n t
(Fig.
e f f e c t was observed at 6 h. DISCUSSION
We have
demonstrated
measurement of i n t r a c e l l u l a r
i n t e r n a l i z e d f l u o r e s c e n t pHi i n d i c a t o r ,
pHi,
in thyroid c e l l s . 347
using
BCECF, an
EGF stimulates c e l l
Vol. 157, No. 1, 1 9 8 8
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
<
z 20
,,.)\ × u E .9
7.4 U C_
Q.
7.3 ~5
2
7.2 I 0
Q
I
~ 1
I
I 20
0
('~
m in
12
~
24
36
48
HOURS
Fig, l , EGF-induced a l k a l i n i z a t i o n in cytoplasmic pH (pHi). BCECF-loaded cells were stimulated by lO nM EGF as indicated by an arrow. Fig. 2. EGF-induced [3HI thymidine incorporation. The cells were incubated with (0-0) or without (e-O) lO nM EGF. EGF stimulated thymidine incorporation significantly at 6 h (P
alkalinization
and
proliferation,
i n d i c a t i n g t h a t Na+/H +
exchange
or
cell
alkalinization
may f u n c t i o n as a transmembrane signal transducer in the action
of EGF in the t h y r o i d c e l l s , EGF has central
b e e n reported to s t i m u l a t e t h y r o i d
problem
t h a t remains to be f u l l y
cell
proliferation
(7-9).
e l u c i d a t e d i s the cascade
of
early
events s t i m u l a t e d by EGF. S t i m u l a t i o n with EGF induces an ordered sequence biochemical transfer
the
proliferation the
events
leading
environmental response?
to c e l l
How does the
i n f o r m a t i o n to the c e l l
The f i r s t
in order
to
elicit
to
signals
earliest
responses to EGF s t i m u l a t i o n i s Na+/H+ exchange or c e l l a l k a l i n i z a t i o n
indicating
that
EGF s t i m u l a t e s c e l l a l k a l i n i z a t i o n
One
to
complex
In t h y r o i d c e l l s ,
DNA s y n t h e s i s .
the
trigger
(I-5).
responses and i n i t i a t e
The i n i t i a l
of
membrane
The EGF-receptor complex i s coupled
e f f e c t o r systems t h a t generate i n t e r n a l s i g n a l s . cellular
plasma
step in the action of EGF i s the binding
EGF receptor at the c e l l surface.
membrane
division.
A
of
and p r o l i f e r a t i o n ,
s t i m u l a t i o n of Na+/H + exchange or a l k a l i n i z a t i o n
may play
important r o l e in t h y r o i d c e l l p r o l i f e r a t i o n .
A v a r i e t y of mitogens have
reported
(I-5),
to induce cytoplasmic a l k a l i n i z a t i o n 348
the
Thus s t i m u l a t i o n of
an been the
Vol. 157, No. 1, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Na+/H+
to be a ubiquitous phenomenon in
exchange
process.
appears
However, t h i s
is the f i r s t
cytoplasmic a l k a l i n i z a t i o n
the
proliferation
report to demonstrate that
in thyroid
EGF induces
cells.
A very important consequence of the activation of Na+/H+ exchange by mitogens is
alkalinization
understanding indicator,
of
of
the
cytoplasm.
A significant
insight
t h i s phenomenon has come from the use of a
BCECF (6).
into
fluorescence
our pH
Changes in cytoplasmic pH can be conveniently monitored
by recording the pH-dependent emission.
Here we show that we can use BCECF to
measure thyroid cell pHi. EGF induces
cytoplasmic a l k a l i n i z a t i o n ,
proliferation,
A major question,
physiological
role
proliferation,
of
which may stimulate
thyroid
to which we have no answer as yet,
alkalinization
in
the
action
of
EGF
is on
cell the cell
Further studies are required to establish whether a l k a l i n i z a t i o n
plays a functional role in the i n i t i a t i o n
of p r o l i f e r a t i o n .
REFERENCES I. Rozengurt, E. (1981) Adv. Enzyme Regul. 19, 61-85i 2. Moolenaar, W.H,, Yarden, Y., de Laat, S.W., and Schlessinger, J. (1982) J. Biol. Chem. 257, 8502-8506. 3. Poussegur, J. (1985) Trends Biochem. Sci. I0, 453-455. 4. Grinstein, S., and Rothstein, A. (1986) J, Membr. Biol. 90, 1-12. 5. Moolenaar, W.H. (1986) Annu, Rev. Physiol. 48, 363-376. 6. Rink, T.J., Tsien, R.Y., and Pozzan, T. (1982) J. Cell Biol. 95, 189-196. 7. Westermark, K., and Westermark, B. (1982) Exp. Cell Res. 138, 47-55. 8. Roger, P.P., and Dumont, J.E. (1982) FEBS Lett. 144, 209-212. 9. Takasu, N., Shimizu, Y., and Yamada, T. (1987) J, Endocrinol. 113, 485-487. I0. Takasu, N., Charrier, B,, Mauchamp, J., and L i s s i t z k y , S. (1978) Eur. J. Biochem. 90, 131-138. I I . Takasu, N., Handa, Y., Shimizu, Y., and Yamada, T. (1984) J. Endocrinol. I01, 189-196.13, 485-487. 12, Thomas, J.A., Buchsbaum, R.N., Zimniak, A., and Racker, E. (1979) Biochemistry 18, 2210-2218.
349
Vol. 157, No. 1, 1988
Pages 346-349
November 30,1988
CYTOPLASMIC pH IN THE ACTION OF EPIDERMAL GROWTH FACTOR (EGF) IN CULTURED PORCINE THYROID CELLS Nobuyuki
Takasu,
Yoshitaka
Nagasawa, Ichiro Yoshifusa Shimizu
Komiya,
Takashi
Yamada and
Department of Gerontology, Endocrinology and Metabolism, School of Medicine, Shinshu University, Asahi 3 - I - I , Matsumoto, Nagano-ken 390, Japan Received
October
20,
1988
We demonstrate measurement of cytoplasmic pH (pHi), using 2 ' , 7 ' - b i s ( 2 carboxyethyl)-5 (and 6-) carboxyfluorescein (BCECF), an internalized fluorescent pHi i n d i c a t o r , in thyroid c e l l s . Using cultured porcine thyr9id cells, we studied the e f f e c t s of epidermal growth f a c t o r (EGF) on pHi and [~H] thymidine incorporation; lO nM EGF a l k a l i n i z e s thyroiQ c@lls and stimulates thymidine incorporation. The r e s u l t s indicate that Na~/HT exchange or c e l l a l k a l i n i z a t i o n may function as a transmembrane signal transducer in the action of EGF in the thyroid c e l l s . ® 1988 Academic P. . . . . I ....
The
mechanisms
proliferation rapidly cells,
by
are
activate
largely
growth factors unknown.
stimulate
Recent evidence suggests
cytoplasmic
pH (pHi) as a p o t e n t i a l
Recently, 2 ' , 7 ' - b i s ( 2 - c a r b o x y e t h y l ) - 5
(BCECF)
(6),
examine
the pHi-regulating mechanisms.
an
proliferation
alkalinization
cell
mitogens
alkalinize
"messenger" for
the cell
has
b e e n used to
Epidermal growth f a c t o r (EGF) is known (7-9).
is unknown. Using cultured porcine thyroid c e l l s , on pHi and thymidine incorporation.
that
and
(and 6-) carboxyfluorescein
i n t e r n a l i z e d fluorescent pHi i n d i c a t o r ,
to stimulate thyroid c e l l
cell
metabolism
Na+/H+ exchange in the plasma membrane and
implicating
growth ( I - 5 ) .
which
However,
the mechanism of t h i s
we studied the e f f e c t s of EGF
We intended to show that Na+/H+ exchange or
might function as a transmembrane signal
transducer
for
thyroid c e l l growth. MATERIALS AND METHODS ! h l r o i d cell c u l t u r e : Thyroid c e l l s were obtained from porcine thyroid gland~ as described previously (lO). Freshly isolated c e l l s were suspended (3xlO v cells/ml) in Eagle's minimum essential medium (MEM) supplemented with 0.5% 0006-291X/88
$1.50
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
346
VoI. 157, No. 1, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
f e t a l - c a l f serum and a n t i b i o t i c s ( p e n i c i l l i n , 200 units/ml; streptomycin, 50 ug/ml). Cells were cultured as a suspension at 370C in a 95% a i r : 5% CO2 water-saturated atmosphere ( I I ) , C~toplasmic ~H ~ H ~ : Cytoplasmic pH (pHi) was measured with the tetraacetoxymethyl ester (BCECF-AM) of 2 ' , 7 ' - b i s ( 2 - c a r b o x y e t h y l ) - 5 (and 6-) carboxyfluorescein (BCECF) (6). PoTcine thyroid c e l l s , cultured f o r 16 h, were washed twice in a standard HEPES-Na~ solution containing 140 mM NaCl, l mM KCI, l mM MgCl~, 1 mM CaCl2, lO mM glucose, and buffered with 20 mM HEPES, pH 7.2. The c e l l { were resuspended in the same b u f f e r containing 3 ~M BCECF-AM and incubated f o r 30 min at 37°C. BCECF-AMwas converted to the impermeant BCECF by cytoplasmic esterases (6). After loading, the c e l l s were washed twice i~ HEPES-Na" solution to remove e x t r a c e l l u l a r dye and were resuspended at 3xlO ~ cells/ml. The BCECF-loaded c e l l s were put into a thermostatic cuvette in a Hitachi F-4000 fluorometer (Hitachi, Tokyo), and the c e l l suspension was continuously s t i r r e d with a magnetic microbar. The pHi-dependent emission i n t e n s i t y was continuously recorded ( e x c i t a t i o n wavelength 500 nm; emission wavelength 530 nm; s l i t s 5.5/ll-nm bandpass). Calibration of cytoplasmic dye fluorescence as a function of pHl was obtained from H e a u ] l l b r a t l o n methods using the K+/H+ lonophore . . . .n l g e r l c l n , which sets [H÷ ] i / [ H + ]o equal to [ K + ] i / [ K + ] o as previously described (12). B r i e f l y , c a l i b r a t i o n was obtained by incubation of BCECF-loaded c e l l s in K~ medium (140 mM KCl, l mM NaCl, l mM MgClg, l mM CaCl~, lO mM glucose) buffered with 20 mM HEPES, and containing nige~icin ( 0 . 7 ~ g ~ l ) . The pHi-dependent emission i n t e n s i t y was then measured. IncorEoration of [~H~ th#midin~: Isolated thyroid c e l l s were cultured f o r 16 h and--then EGF and O : l ~ C i - o f - [ H] thymidine were added to measure the e f f e c t of EGF on thymidine incorporation. After incubation, the c e l l s were collected and washed three times with lOZ t r i c h l o r o a c e t i c acid (TCA) and dissolved in 0.3 N NaOH. T h e n t h e i r r a d i o a c t i v i t i e s were measured in a l i q u i d scintillation counter. Materials e t c . : Murine epidermal growth f a c t o r (EGF) was obtained from Collaborative Research (Waltman, MA, U.S.A.); n i g e r i c i n and HEPES from Sigma (St. Louis, MO, U.S.A.); fetal c a l f s~rum and Eagle's MEM from Flow Laboratories ( I r v i n e , Scotland, U.K.); [~H] thymidine (2 Ci/mmol) from New England Nuclear Corp. (Boston, MA, U.S.A.); BCECF-AMfrom Molecular Probes, Inc. (Junction City, OR, U.S.A.). BCECF-AM was dissolved in dimethyl s u l f o x i d e and stored at -20°C. All other chemicals were of the highest p u r i t y available commercially. Experiments were conducted at least 4 times. Typical data and the final concentration of EGF are shown in the t e x t and the f i g u r e . Data were s t a t i s t i c a l l y analyzed with analysis of variance (ANOVA). P
-
-
t
.
-
•
RESULTS EGF-stimulated thyroid c e l l a l k a l i n i z a t i o n and ~ m ~
~Q£~[~9 r a t i ° n ,
The c e l l s were loaded with BCECF and then pHi was recorded continuously (Fig. I).
The resting pHi of porcine thyroid c e l l s in HEPES-Na+ solution at 37°C was
7.22+0.04
(n=8).
alkalinization,
Addition
of lO nM EGF to these c e l l s induced
intracellular
reaching a pH of 7.39+0.06 (n=8) in 3 min.
Ten nM EGF produced a gradual increase in [3H] thymidine incorporation 2). A s i g n i f i c a n t
(Fig.
e f f e c t was observed at 6 h. DISCUSSION
We have
demonstrated
measurement of i n t r a c e l l u l a r
i n t e r n a l i z e d f l u o r e s c e n t pHi i n d i c a t o r ,
pHi,
in thyroid c e l l s . 347
using
BCECF, an
EGF stimulates c e l l
Vol. 157, No. 1, 1 9 8 8
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
<
z 20
,,.)\ × u E .9
7.4 U C_
Q.
7.3 ~5
2
7.2 I 0
Q
I
~ 1
I
I 20
0
('~
m in
12
~
24
36
48
HOURS
Fig, l , EGF-induced a l k a l i n i z a t i o n in cytoplasmic pH (pHi). BCECF-loaded cells were stimulated by lO nM EGF as indicated by an arrow. Fig. 2. EGF-induced [3HI thymidine incorporation. The cells were incubated with (0-0) or without (e-O) lO nM EGF. EGF stimulated thymidine incorporation significantly at 6 h (P
alkalinization
and
proliferation,
i n d i c a t i n g t h a t Na+/H +
exchange
or
cell
alkalinization
may f u n c t i o n as a transmembrane signal transducer in the action
of EGF in the t h y r o i d c e l l s , EGF has central
b e e n reported to s t i m u l a t e t h y r o i d
problem
t h a t remains to be f u l l y
cell
proliferation
(7-9).
e l u c i d a t e d i s the cascade
of
early
events s t i m u l a t e d by EGF. S t i m u l a t i o n with EGF induces an ordered sequence biochemical transfer
the
proliferation the
events
leading
environmental response?
to c e l l
How does the
i n f o r m a t i o n to the c e l l
The f i r s t
in order
to
elicit
to
signals
earliest
responses to EGF s t i m u l a t i o n i s Na+/H+ exchange or c e l l a l k a l i n i z a t i o n
indicating
that
EGF s t i m u l a t e s c e l l a l k a l i n i z a t i o n
One
to
complex
In t h y r o i d c e l l s ,
DNA s y n t h e s i s .
the
trigger
(I-5).
responses and i n i t i a t e
The i n i t i a l
of
membrane
The EGF-receptor complex i s coupled
e f f e c t o r systems t h a t generate i n t e r n a l s i g n a l s . cellular
plasma
step in the action of EGF i s the binding
EGF receptor at the c e l l surface.
membrane
division.
A
of
and p r o l i f e r a t i o n ,
s t i m u l a t i o n of Na+/H + exchange or a l k a l i n i z a t i o n
may play
important r o l e in t h y r o i d c e l l p r o l i f e r a t i o n .
A v a r i e t y of mitogens have
reported
(I-5),
to induce cytoplasmic a l k a l i n i z a t i o n 348
the
Thus s t i m u l a t i o n of
an been the
Vol. 157, No. 1, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Na+/H+
to be a ubiquitous phenomenon in
exchange
process.
appears
However, t h i s
is the f i r s t
cytoplasmic a l k a l i n i z a t i o n
the
proliferation
report to demonstrate that
in thyroid
EGF induces
cells.
A very important consequence of the activation of Na+/H+ exchange by mitogens is
alkalinization
understanding indicator,
of
of
the
cytoplasm.
A significant
insight
t h i s phenomenon has come from the use of a
BCECF (6).
into
fluorescence
our pH
Changes in cytoplasmic pH can be conveniently monitored
by recording the pH-dependent emission.
Here we show that we can use BCECF to
measure thyroid cell pHi. EGF induces
cytoplasmic a l k a l i n i z a t i o n ,
proliferation,
A major question,
physiological
role
proliferation,
of
which may stimulate
thyroid
to which we have no answer as yet,
alkalinization
in
the
action
of
EGF
is on
cell the cell
Further studies are required to establish whether a l k a l i n i z a t i o n
plays a functional role in the i n i t i a t i o n
of p r o l i f e r a t i o n .
REFERENCES I. Rozengurt, E. (1981) Adv. Enzyme Regul. 19, 61-85i 2. Moolenaar, W.H,, Yarden, Y., de Laat, S.W., and Schlessinger, J. (1982) J. Biol. Chem. 257, 8502-8506. 3. Poussegur, J. (1985) Trends Biochem. Sci. I0, 453-455. 4. Grinstein, S., and Rothstein, A. (1986) J, Membr. Biol. 90, 1-12. 5. Moolenaar, W.H. (1986) Annu, Rev. Physiol. 48, 363-376. 6. Rink, T.J., Tsien, R.Y., and Pozzan, T. (1982) J. Cell Biol. 95, 189-196. 7. Westermark, K., and Westermark, B. (1982) Exp. Cell Res. 138, 47-55. 8. Roger, P.P., and Dumont, J.E. (1982) FEBS Lett. 144, 209-212. 9. Takasu, N., Shimizu, Y., and Yamada, T. (1987) J, Endocrinol. 113, 485-487. I0. Takasu, N., Charrier, B,, Mauchamp, J., and L i s s i t z k y , S. (1978) Eur. J. Biochem. 90, 131-138. I I . Takasu, N., Handa, Y., Shimizu, Y., and Yamada, T. (1984) J. Endocrinol. I01, 189-196.13, 485-487. 12, Thomas, J.A., Buchsbaum, R.N., Zimniak, A., and Racker, E. (1979) Biochemistry 18, 2210-2218.
349