Vol. 187, No. 1,1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 529-536
August 31,1992
AUGMENTATION OF TRANSIENT LOW-TIIRESIIOLD Ca 2+ CURRENT INDUCED BY GTP-BINDING PROTEIN SIGNAL TRANSDUCTION SYSTEM IN GH3 PITUITARY CELLS
NOBUYUKI SUZUKI I * , IIIROSIII TAKAGI 2 , TOIIRU YOSHIOKA 2 AKItlIRO TANAKADATE3 , and~IASAAI(IRA KANO1
iDepartment
of Physiology
and
3Facility
of Nedical
Engineering,
S~hool of Medicine, Kitasato University, Sagamihara, Kanagawa 228, Japan ~Department of Basic Sciences, School of Human Sciences, Waseda University Tokorozawa, Saitama 359, Japan
Received July 23, 1992
SUI~¥ We characterized the effects of thyrotropin-releasing hormone (TRH; 500 n~) and guanosine 5'-O-3-thiotriphosphate (GTP~S; 50 ~M) on two types of Ca z+ currents in pituitary-hormone-secretory GH 3 cells and were surprised to find marked increases in transient, low-threshold Ca z+ currents (T currents) induced by extracellularly applied TRH or intracellularly applied GTPTS. The effect of TRH was blocked by intracellularly applied guanosine 5'-O-2-thiodiphosphate (GDP~S; i00 ~M). The increase in the T current was found to be accompanied by a decrease in long-lasting, high-threshold Ca 2+ current (L-current), in response to both TRIÂ or GTPTS. These indicate that the enhancement of Ca 2+ influx by TRII (500 nM) is largely conferred by T currents in GH 3 cells. A reduced concentration of TRII (5 nM) still markedly increased the T current, but failed to decrease the L current. These data suggest that the augmentation of the T currents as well as depression of the L currents by TRH (500 nM), through the activation of a GTP-binding protein, may constitute an important regulatory mechanism of sustained pituitary hormone secretion in GH 3 cells. ® 1992 Academic press, Inc.
The hormone
modulation (TRH),
crease
in
tained
hormone
currents
Ca 2+
be
cytosolic
an
free
secretion
by TRH
Previous transient
of Ca 2+ channels
may
low-threshold
channels
Ca 2+
(6-8),
as well
studies
not
of
thyrotropin-releasing
regulating
([Ca2+]i),
as inhibition
which
the
sustained
results
in
insus-
of voltage-dependent
K+
(4,5).
(T-type)but
the neuropeptide
mechanism
concentration
(i-3),
in GH 3 cells
whole-cell
by
important
of
in GH 3 cells and the
demonstrated
long-lasting transient
the
high-threshold
high-threshold
presence
of
(L-type)-,
(N)-type.
T-
*To whom correspondence should be addressed. Abbreviations used; GTPTS, guanosine 5'-0-3-thiotriphosphate;^GTP, guanosine te-~GDP~S, guanosine 5'-0-2-thiodiphosphate; [CaZ+]$, cytosolic free Ca2 concentration; T current, transient low-threshold Ca2 current; L current, long-lasting high-threshold Ca2+ current.
~
529
0006-291 X/92 $4.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 187, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and L- channels function very d i f f e r e n t l y in the conversion of t i a l s to i n t r a c e l l u l a r calcium transients, (9).
The individual roles of
Ca2+ channel
action poten-
as evidenced by McCobb and Beam
the two d i s t i n c t types
of voltage-dependent
in the specialized hormonal function in GH3 c e l l s remain to be
determined. We have recently suggested that the sustained elevation of [Ca2+]i t r i g gered by TRH in GH3 c e l l s might be due to r e p e t i t i v e openings of T-type Ca2+ channels causing a series of transient Ca2+ influx largely through T-channels (10),
as a result of the enhancement of action potential f i r i n g over a pro-
longed period induced by TRH e f f e c t in GH3 c e l l s (4, 11-14; for a review, see 15).
Also, TRH inhibited L-type Ca2+ current has been c l e a r l y demonstrated by
Levitan and Kramer (8) and Kramer et al. patch clamp technique. through T-type
(16), using the whole-cell perforated
This r e s u l t f u r t h e r suggests that
Ca2+ i n f l u x
Ca2+ channels is responsible for the sustained increase of
[Ca2~] i . In t h i s w h o l e - c e l l voltage-clamp study, we concentrated on examining whether TRH modulates the T-type and
L-type currents in GH3 c e l l s ,
through
the activation of a G protein, which may regulate the frequency of Ca2+ action p o t e n t i a l s , thereby controlling the level of the sustained
[Ca2+]i in GH3
cells.
MATERIALS AND I4ETIIODS Cell culture A cloned line of rat pituitary tumor cells, GII 3 cells, was purchased from American Type Culture Collection (Rockville, MD) through Dainihon-Seiyaku (Oosaka, Japan), and the cells were maintained in Ham's F-IO medium supplemented with 15% horse serum, 2.5% fetal bovine serum under 5% C02/ air at 37 °C (17). The medium contained 50 units/ ml penicillin and 50 g/ ml streptomycin. Cells were plated onto glass coverslips (15 mm diameter) and electrophysiological experiments were performed 2-7 days later. Physiological recording The whole-cell patch-clamp technique (18) was used to examine cells in a bath perfused (i ml/min) at room temperature. The patch electrode resistance ranged from 3 to 8 MD when filled with internal solutions. A model S-3666 patch clamp whole cell clamp amplifier (Nihon Kohden, Tokyo) with a type P-10G probe (feedback resistor: i0 GD) was used. Voltage pulses and currents were sampled online at 200 ~sec intervals with a computer (VM-2, NEC, Japan). Whole cell current measured under voltage clamp conditions was sampled after filtered (+3 db at 3 kHz). The stimulation rate was 0.5 Hz, Linear leak and capacitance currents were subtracted digitally. Ca 2+ currents were recorded with a pipete filled with an intracellular solution containing 145 mM Cs aspartate, 5 mM EGTA A 2 mM Mg-ATP, i0 mM HEPES (pH 7.4); cells were continuously perfused with CaZ+-TEA saline consisting of 2 mM CaCl 2, 138 mM TEA-C1, 2 mM MgCl 2, 20 mM HEPES (pH 7.3 was adjusted by NaOH). Tetrodotoxin (TTX) at a concentration of 500 nM was added to suppress currents through Na + channels. TRH (Sigma) in distilled water was dissolved in the perfusing saline. Nonhydrolyzable GTP analog, guanosine 5'-O-3-thiotriphosphate (GTPTS; Boehringer-Mannheim), an activator of G protein (19), was added to the patch pipet solution at a concentration of 20 ~M in Figs. 1 and 3. In Fig. 4B, GTPTS was added to the patch pipete solution at 50 ~M. In some experiments, hydrolyzable guanosine 5'-triphosphate (GTP, BoehringerMannheim) was added to the patch pipete solution at 20 ~M. Nonhydrolyzab]e
530
Vol. 187, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
GDP a n a l o g , g u a n o s i n e 5 ' - O - 3 - t h i o t r i p h o s p h a t e (GDPflS; B o e h r i n g e r - M a n n h e i m ) , an i n a c t i v a t e r o f G p r o t e i n (19), was added to t h e p a t c h p i p e t s o l u t i o n a t a concentration o f 100 ZM i n F i g . 2. S o l u t i o n c h a n g e s i n t h e c h a m b e r w e r e c o m p l e t e d w i t h i n 3 min. O th e r c h e m i c a l s were from Wako Pure Chem. (Oosaka, Japan).
RESULTS T- a n d L - c h a n n e l s
F i g u r e 1A ( c o n t r o l mY,
depolarizing
above
test
each t r a c e s )
traces)
tionally
L-type
msec t e s t
pulses
associated
(B),
(for
an example,
-20 and -10 mV inactivation
s e e t h e arrow heads
relations
(C) and T c u r r e n t
To i s o l a t e
current
slow
rapid inactivation
components were p r e s u m a b l y f u l l y
current-voltage
L current
circles).
pulses at
showing v e r y
o f -80 indicated with
produced
during
addi-
maintained
The a m p l i t u d e of L - c u r r e n t was measured a t t h e end o f 160
time the T-current the
T - t y p e c u r r e n t s showing
and t e s t
currents
from a h o l d i n g p o t e n t i a l
t o between -50 and -30 mV ( p o t e n t i a l
induced
depolarization.
shows t h a t ,
pulses
maintained depolarization,
i n GH3 c e l l s .
the
experiments of Figs. at a concentration
(D),
amplitude
(C) was s u b t r a c t e d
the
measured
peak
in
amplitude
GTPTS
of
1A),
(D),
at
which
In F i g .
(= T + L)
control
of T c u r r e n t
from t h e
1 and 3,
depict
in Fig.
inactivated.
current
solution
the
( o p en
amplitude
peak c u r r e n t
was added t o t h e p a t c h
(B).
of L In
pipet
o f 20 zM, a l t h o u g h we c o u l d n o t know a c t u a l
1,
the
solution
concentrations
o f GTP7 S r e n d e r e d w i t h i n c e l l s . Effects
o f TRH {500 n~) on T- and L - c u r r e n t s .
F i g u r e 1A (TRH t r a c e s ) T current
amplitude
duced by TRH appreciable
shows t h a t TRH (500 nM) d r a m a t i c a l l y
i n GH3 c e l l s .
was s e e n o v e r
A significant
test
potential
In c o n t r a s t
to t h e i n c r e a s e
1A (TRH t r a c e s )
illustrates
amplitude
current,
as
amplitude
of L c u r r e n t
decrease
of L in
the
that
1C; s o l i d
shows
decrease
that
both this
were a p p r e c i a b l y Figs.
reversed
1C and 1D),
" r u n down" effect
at
to
test
pulses
by TRH
and
the
the
at
of r e c o v e r y ,
the
- 1 0 mV.
The
test
potentials
1A ( R e c o v e r y t r a c e s ) increase
in T c u r r e n t
( s e e a l s o open t r i a n g l e s
t h e d e c r e a s e of L c u r r e n t
extent
but
rather
in
was n o t c a u s e d by by a d e p r e s s i v e
o f TRH.
S u p e r i m p o s e d Ca 2+ c u r r e n t
traces
in F i g .
2 were measured
in the
i n c l u d i n g GDP a n a l o g GDP~S (100 ZM) i n t r o d u c e d w i t h a p a t c h p i p e t , tivator
Figure
decreased
-20 and
Figure
in L c u r r e n t
that
of
pro-
1D; s o l i d
of T c u r r e n t ,
was seen
circles).
when TRH was washed out
indicating
least
in t h e a m p l i t u d e
the
-10 mV, w i t h no
of about -60 mV ( F i g .
TRH (500 nM) d r a m a t i c a l l y
shown a t
e x c e e d i n g -20 mV ( F i g .
of T c u r r e n t
r an g e of -50 t o
change i n t h e t h r e s h o l d p o t e n t i a l
circles).
increase
increased
of G p r o t e i n
no s i g n i f i c a n t
(19).
changes
in
In F i g . the
2B, t h e a d d i t i o n
T currents
531
cell
an i n a c -
o f TRH (500 nM) p r o d u c e d
(compare B w i t h
A),
although
some
V o l . 1 8 7 , N o . 1, 1 9 9 2
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
A -50
-40
-30
-20
T
-10
T
-5O
-40
-30
-20
-10
-50
-40
-30
-20
-10
Control
TRH
Recovery
B
C mV 20
-89_, .~ -4.0 ,
~
D
- 8 9 . . . , .. - ~ o
~.
mV o 29
-80
,.~ - 4 0
mV i
20 I
10(
pA -300
~
~00
pA
500
pA
F i g . 1. TRIt (500 nM) i n c r e a s e d T - t y p e Ca 2+ c h a n n e l c u r r e n t , b u t r e d u c e d Lt y p e Ca 2+ c h a n n e l c u r r e n t . Currents were recorded under whole-cell v o l t a g e - c l a m p i n GH3 c e l l s . I n (A), Ca 2+ c u r r e n t t r a c e s were o b t a i n e d j u s t before (Control traces), d u r i n g a p p l i c a t i o n of 500 nM TRH (TRH t r a c e s ) and f o l l o w i n g r e c o v e r y o f 5 min ( R e c o v e r y t r a c e s ) , t o t h e same GH3 c e l l . From a holding potential of -80 mV, d e p o l a r i z i n g p u l s e s (160 msec d u r a t i o n ) t o b e tween -50 and -30 mV, t h e p o t e n t i a l i n d i c a t e d above each t r a c e s , i n d u c e d Tt y p e c u r r e n t , and t e s t p u l s e s a t -20 and -10 mV i n (A) and -20 and +20 mV i n (B) p r o d u c e d L - t y p e c u r r e n t , t o g e t h e r with T-type current. The a m p l i t u d e o f L - t y p e c u r r e n t was m e a s u r e d a t t h e end o f 160 msec t e s t p u l s e s ( s e e t h e a r r o w heads in Control traces). The a s s o c i a t e d c u r r e n t - v o l t a g e r e l a t i o n s d e p i c t t h e peak (= T - t y p e + L - t y p e ) c u r r e n t (B), L - t y p e c u r r e n t (C) and T - t y p e c u r r e n t (D), m e a s u r e d i n c o n t r o l s o l u t i o n (open c i r c l e s ) , i n t h e p r e s e n c e o f 500 nM TRH ( s o l i d circles), and f o l l o w i n g r e c o v e r y a t 5 min ( o p e n t r i a n g l e s ) . C u r r e n t t r a c e s h a v e been c o r r e c t e d f o r l i n e a r l e a k and c a p a c i t a n c e c u r r e n t i n t h l s and s u c c e e d i n g f i g u r e s . I n f i g u r e s 1 and 3, G T P r S was added t o t h e p a t c h p i p e t s o l u t i o n a t a c o n c e n t r a t i o n o f 20 zM, a l t h o u g h we c o u l d n o t know the actual concentrations o f GTPTS w i t h i n c e l l s . Throughout this e x R e r i m e n t , GH3 c e l l s were b a t h e d in N a + - f r e e , Ca2+-TEA s o l u t i o n t o i s o l a t e Ca c u r r e n t s , and TTX was added t o s u p p r e s s c u r r e n t t h r o u g h Na + c h a n n e l s .
Z+
increases in the L currents were observed (arrow),
i n d i c a t i n g t h a t the cyto-
s o l i c GDP~S markedly blocked the e f f e c t of TRH (500 nM) to produce both an increase of T current and a decrease of L current as seen in Fig. corresponds to the control experiment of Fig. i . 532
1.
This
Vol. 187, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A
B
F i g . 2. C y t o s o l i c GDPflS, an i n a c t i v a t o r of G p r o t e i n , b l o c k e d t h e e f f e c t of TRH as s e e n in Fig. 1. GDPBS (100 ZM) was r e n d e r e d w i t h i n t h e c e l l v i a a p a t c h p i p e t in (A) and (B). Superimposed Ca2+ c u r r e n t t r a c e s in (A) and (B) were evoked w i t h t e s t p u l s e s (160 msec d u r a t i o n ) spaced by 10 mV t o between -SO and -20 mY, and +10 mV ( a r r o w s ) from a h o l d i n g p o t e n t i a l o f -80 mY. Note t h a t i n t h e p r e s e n c e o f t h e c y t o s o l i c GDPflS, T c u r r e n t s in c o n t r o l t r a c e s (A) r e m a i n e d l a r g e l y u n c h a n g e d by t h e a d d i t i o n o f SO0 nM TRH (B), a l t h o u g h some i n c r e a s e in the L - c u r r e n t was o b s e r v e d (B, a r r o w ) . V e r t i c a l and h o r i z o n t a l c a l i b r a t i o n b a r s r e p r e s e n t 50 pA and 40 msec, r e s p e c t i v e l y .
Effect In Fig.
3,
superimposed (arrows), currents
of a lowered concentration control on c u r r e n t
with
o f - 8 0 mV.
traces
t a k e n 30 s e e a f t e r
pulse
T currents
increases
by
in
marked w i t h
(Fig.
1).
S.E.M.;
N=6)
at
The the
of T current
the
test
increase test
pulses
at
-20
of
-40
threshold
-30
to and
traces -30
10
o f 5 nM TRtt show
mY a n d mY,
shown
pure T
L currents
from
a
holding
o f TRft (5 nM) p r o d u c e d s i g n i f similar was
to those by
- 3 0 mY. at
are
the application
-50
T current
potential
by TRH (5 nM) o c c u r r e d
v -50
of
of T current,
- 4 0 a n d - 1 0 mY, w i t h no c h a n g e i n t h e
,
range
of
open triangles,
Control current
k reduced concentration
in the amplitude
nM TRH
crease
traces
evoked by t e s t
potential icant
current
m e a s u r e d f r o m t h e same c e l l .
together
o f T~I (5 nM) on T c u r r e n t s .
test
35 The
potentials
potential
-20
i n d u c e d b y 500
± 6.0%
(mean
appreciable in the
of about
range of
- 6 0 mY and
+10 -
_
~I00 pA 50 ms Fig. 3.
A lowered c o n c e n t r a t i o n of TRH (S nM) markedly i n c r e a s e d t h e a m p l i tude of t h e T c u r r e n t , whereas no a p p r e c i a b l e change o f t h e L c u r r e n t was seen. C o n t r o l c u r r e n t t r a c e s marked with open t r i a n g l e s a r e shown s u p e r i m p o s e d on c u r r e n t t r a c e s taken 30 sec a f t e r the a p p l i c a t i o n of TRH ( a r r o w s ) , in t h e same c e l l . From a h o l d i n g p o t e n t i a l o f - 8 0 mV, t e s t p u l s e s to between -SO and -30 mV as i n d i c a t e d above each t r a c e induced T-type c u r r e n t , and t e s t p u l s e s a t -20 and 10 mV produced both L-type c u r r e n t and T - t y p e c u r rent. Six of 7 c e l l s examined showed s i g n i f i c a n t i n c r e a s e s o f t h e T c u r r e n t s by 35 + 6.0% (mean ± S.E.M.; N=6) at the test potential of -30 mV i n r e s p o n s e t o TRH a p p l i c a t i o n . 533
±
in-
Vol. 187, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A
B
F i g . 4. I n t r a c e l l u l a r l y a p p l i e d GTPTS with a p a t c h p i p e t c o n t a i n i n g 50 ~M GTPrS induced b o t h an i n c r e a s e of T c u r r e n t and a d e c r e a s e o f L c u r r e n t in (B}, t h r o u g h t h e a c t i v a t i o n of a G p r o t e i n , and hence mimicked t h e e f f e c t s of e x t r a c e l l u l a r l y a p p l i e d TRH (500 nM) shown in Fig. 1. F a m i l i e s of s u p e r i m p o s e d Ca2+ c u r r e n t t r a c e s were o b t a i n e d from a c e l l w i t h o u t GTPTS (A) and a c e l l w i t h GTP~S ( B ) . No TRH was p r e s e n t e x t r a c e l l u l a r l y . C u r r e n t s were evoked w i t h t e s t p u l s e s (160 msec d u r a t i o n ) spaced by 10 mV to between -50 and +10 mV f o r (A) and (B) from a h o l d i n g p o t e n t i a l of -80 mV. Note t h a t the Ca 2+ c h a n n e l c u r r e n t was l a r g e l y i n a c t i v a t i n g T c u r r e n t i n (B). (A) and (B) show d a t a from d i f f e r e n t c e l l s . The mean r a t i o o f t h e a m p l i t u d e s of T c u r r e n t measured a t a t e s t p o t e n t i a l of -20 mV r e l a t i v e t o t h e a m p l i t u d e s of L c u r r e n t a t 10 mV was 2.4 (N=3) in a c e l l with 50 Z M GTP7 S, in c o n t r a s t to t h e r e d u c e d mean r a t i o of 1,0 in a c e l l w i t h o u t GTPTS (N=4, d a t a not shown). V e r t i c a l and h o r i z o n t a l c a l i b r a t i o n b a r s r e p r e s e n t 50 pA and 40 msec, r e s p e c t i v e l y , f o r (A) and (B). A broken l i n e in (B) i n d i c a t e s 0 c u r r e n t l e v e l .
the
peak
position
concentration current,
of
of
about
as s e e n at
a test
When h y d r o l y z a b l e GTP~S,
rents
5 min a f t e r
within
potential
~iEicked
from a cell
respectively.
The
of
produced both
current
- 8 0 mV.
not
applied
test
traces
internally
with
of
were
elicited
increments
a patch
lowered of
pipet
of non-
increase
of T
- 3 0 mY.
by
with
test
Ca 2+
z M),
in
the
from a holding
TRH, 50 ZM GTP7 S
of L current
potentials
(50
potentials
(160 msec d u r a t i o n )
of both
concentration
current
GTP 7 S
of
(Fig.
currents GTPTS
was f o u n d t o m i m i c t h e
effects
by externally
TRH
applied
4B), (N=3,
(50
ZM)
on t h e
T- a n d L - t y p e
Ca 2+ c u r r e n t s
induced
These suggest
that
o f TRH (500 nM) i s m e d i a t e d b y a G p r o t e i n .
the effect
L
on t h e T c u r -
This of
superimposed
and a d e c r e a s e
elevated
instead
effect
the absence of extracellular
the
this
amplitude
cells,
similar
GTP7 S and a c e l l
of T current
Hence,
the
o f T~I on T- a n d L - c u r r e n t s .
change in the threshold
shown).
within
potential
representative without
Despite
an i n c r e a s e
w i t h no s i g n i f i c a n t
However, in
o f t h e membrane s e a l . the
the effects
r a n g e o f - 5 0 t o +10 mV i n 10 mV potential
shown). a change
o f 10 mV.
the rapture
4A a n d 4B s h o w
obtained
not
induce
zM) was r e n d e r e d
was b y 27 + 2.0% (N=3) a t
Figures
data
to
TRH (5 nM) a p p e a r e d t o g i v e
GTP7 S (50 ~ )
traces
mV ( d a t a
GTP (20
hydrolyzable
current
-20
TRH (5 nM) f a i l e d
(500
nM}.
DISCUSSION The p r e s e n t significantly
experiments
revealed
augments T-current
for
amplitude, 534
the
first
time
that
TRH (500
through the activation
riM)
of a G pro-
Vol. 187, No. 1, 1992
tein,
in
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
GH3 c e l l s
especially
under
significant
whole-cell
such as
the
enhancement o f t h e f r e q u e n c y of Ca 2+ a c t i o n p o t e n t i a l s
is
increasing
and s u s t a i n i n g
secretion current
a secreting
cell
The i n c r e a s e
we o b s e r v e d i n t h i s
potential
frequencies
s t u d y may r e s u l t
i n GH3 c e l l s ,
d e p r e s s i o n o f K+ c u r r e n t s by TRH ( 4 , 5 ) . mimicked by i n t r a c e l l u l a r l y a p p l i e d GDP~S,
fact
is
i n which
a prerequisite
for
i n t o n i c hormone
in t h e a m p l i t u d e
of T
t h e enhancement o f Ca 2+
possibly
This e f f e c t
concomitant
with
the
o f TRtt on T c u r r e n t
was
a p p l i e d GTP 7 S and was b l o c k e d by i n t r a c e l l u l a r l y
suggesting
d u c t i o n mechanism t h a t
in
This
Gtt3 c e l l ,
t h e l e v e l of [Ca2+]i, which r e s u l t s
( f o r a r e v i e w s e e 15).
that
action
for
voltage-clamp conditions.
that
links
a G protein
TRH r e c e p t o r
is
i n v o l v e d in the
activation
signal
trans-
to the modulation
of T-
t y p e Ca 2+ c h a n n e l s . The i n c r e a s e reversible riM).
in T-current
amplitude
This
T-current
reduction
in L - c u r r e n t
amplitude,
makes i t
prolonged enhanced current.
is
possible
Ca 2+ l e v e l
action potentials [Ca2+]i
is
that
the
and
channel activity
Kramer
(8)
i n which t h e v e s i c l e
retains
indicating
cell
of
or o u t - s i d e
have a p p a r e n t l y
(4,20).
The p r e s e n t
configuration
have is
the
tained
result
using patch pipet
increase
frequencies relatively
largely influx
T is
the accumulated frequency
an a c c u r a t e of
in
during the
calcium
increased
of
regulation
Ca 2+ e n t r y
largely
u n i f o r m and i n s e n s i t i v e
recently
shown t h a t inhibited
named t h e
"perforated
out
patches
that
TRH has
L currents
successfully solution
supplemented
vesicle
method",
in
an o u t -
L-channel conductance; In c o n v e n t i o n a l
compo-
Ca 2+ c h a n n e l s
in conventional
by TRH (500 nM), with
the
whole-
cytoplasmic
on L t y p e
recorded
inhibited
Ca 2÷
decreased the probability
in which c r u c i a l no e f f e c t
L-type
by TRtt (100 nM),
ion channels
t h a t TRH r e v e r s i b l y single
recorded L cur-
single
dramatically
c y t o p l a s m and c o n t a i n s
been l o s t ,
may be
of
but t h a t
c h a n n e l c o n d u c t a n c e i s 32 pS i n 110 mM b ar i u m .
recordings
nents
cell
amount
Furthermore,
o f a L c h a n n e l b e i n g open w i t h o u t a f f e c t i n g single
the
i n d u c e d by TRH i s
findings using whole-cell
clamp c o n f i g u r a t i o n
s i d e out o r i e n t a t i o n ,
to
TRH (500
waveform (9).
from GH3 c e l l s
u s i n g a new p a t c h
net
because
whose amount may be
in a c t i o n p o t e n t i a l
Levitan
firing
by c h a n g i n g
In accordance with the present rents,
contrast
t h e Ca 2÷ c h a n n e l c u r r e n t
as s u g g e s t e d by (16).
through T channels,
in
in response to
of L - t y p e c u r r e n t ,
increased
c o u l d be p o s s i b l e
to alteration
that
Ca 2+ a c t i o n p o t e n t i a l
Hence i t
intracellular
amplitude
likely
reduced because of the i n h i b i t i o n
of
was f o u n d t o be a c c o m p a n i e d by a
d e c r e a s e i n L - t y p e Ca 2+ c u r r e n t a m p l i t u d e
GTPTS
wholewas
(20
obZM).
ACKNOWLEDGr~ENTS
We t h a n k D r s . H. R o b i n s o n and C . - F , Wu f o r r e a d i n g t h e m a n u s c r i p t . T h i s work was s u p p o r t e d by a G r a n t - i n - A i d (03680223) f o r S c i e n t i f i c Research t o N.S. from t h e M i n i s t r y o f E d u c a t i o n , S c i e n c e , and C u l t u r e o f J a p a n . 535
Vol. 187, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES
i. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
Albert, P.R., and Tashjian, A.H.Jr. (1984a) J. Biol. Chem., 259: 5827-5832. Albert, P.R., and Tashjian, A.H.Jr. (1984b) J. Biol. Chem., 259: 15350-15363. Gershengorn,M.C., and Thaw, C. (1985) Endocrinology, 116:591-596. Dubinsky, J. M., and Oxford, G. M. (1985) P r o c . Natl. Acad. Sci.U.S.A., 82:4282-4286. B a u e r , C.K., Meyerhof, W., and Schwarz, J.R. (1990) J. Physiol., 429:169-189. Matteson, D.R., and Armstrong, C.M. (1986) J. Gen. Physiol., 87:161-182. Simasko,S.M., Weiland, G.A., and Oswald, R.E. (1988) Am. J. Physiol., 254: E328-E336. Levitan, E.S., and Kramer, R.H. (1990) Nature, 348:545-547. McCobb,D.P., and Beam, K.G. (1991) Neuron, 7:119-127. Suzuki, N., Kudo, Y., Takagi, H., Yoshioka, T., Tanakadate, A., and Kano, M. (1990) J. Cell. Physiol., 144:62-68. Kidokoro, Y. (1975) Nature, 258:741-742. Dufy, B., Vincent, J.D., Fleury, H., Pasquier, P., Gourdji, D., and Vidal, A.T. ( 1 9 7 9 ) Science, 204:509-511. Ozawa, S., and Kimura, N. (1979) P r o c . Natl. Acad. Sci. U.S.A., 76:6017-6020. Benham, C.D. (1989) J. Physiol., 415:143-158. Ozawa, S., and Sand, O. (1986) Physiol. Rev., 66:887-952. Kramer, R.H., Kaczmarek, L.K., and Levitan, E . S . (1991) Neuron, 6:557-563. Tashjian, A,H.Jr., Yasumura, Y., Levine, L., Sato, G.H., and Parker, M.L. ( 1 9 6 8 ) Endocrinology, 82:342-352. Marty, A., and Neher, E. (1983) In Single Channel Recording (Sakmann, B., and Neher, E.,eds.) pp.107-121, Plenum, New York. Gilman,A.G. (1987) Ann. Rev. Biochem., 56:615-649. Dufy, B., Jaken, S., and Barker, J.L. (1987) Endocrinology, 121:793-802.
536
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 529-536
August 31,1992
AUGMENTATION OF TRANSIENT LOW-TIIRESIIOLD Ca 2+ CURRENT INDUCED BY GTP-BINDING PROTEIN SIGNAL TRANSDUCTION SYSTEM IN GH3 PITUITARY CELLS
NOBUYUKI SUZUKI I * , IIIROSIII TAKAGI 2 , TOIIRU YOSHIOKA 2 AKItlIRO TANAKADATE3 , and~IASAAI(IRA KANO1
iDepartment
of Physiology
and
3Facility
of Nedical
Engineering,
S~hool of Medicine, Kitasato University, Sagamihara, Kanagawa 228, Japan ~Department of Basic Sciences, School of Human Sciences, Waseda University Tokorozawa, Saitama 359, Japan
Received July 23, 1992
SUI~¥ We characterized the effects of thyrotropin-releasing hormone (TRH; 500 n~) and guanosine 5'-O-3-thiotriphosphate (GTP~S; 50 ~M) on two types of Ca z+ currents in pituitary-hormone-secretory GH 3 cells and were surprised to find marked increases in transient, low-threshold Ca z+ currents (T currents) induced by extracellularly applied TRH or intracellularly applied GTPTS. The effect of TRH was blocked by intracellularly applied guanosine 5'-O-2-thiodiphosphate (GDP~S; i00 ~M). The increase in the T current was found to be accompanied by a decrease in long-lasting, high-threshold Ca 2+ current (L-current), in response to both TRIÂ or GTPTS. These indicate that the enhancement of Ca 2+ influx by TRII (500 nM) is largely conferred by T currents in GH 3 cells. A reduced concentration of TRII (5 nM) still markedly increased the T current, but failed to decrease the L current. These data suggest that the augmentation of the T currents as well as depression of the L currents by TRH (500 nM), through the activation of a GTP-binding protein, may constitute an important regulatory mechanism of sustained pituitary hormone secretion in GH 3 cells. ® 1992 Academic press, Inc.
The hormone
modulation (TRH),
crease
in
tained
hormone
currents
Ca 2+
be
cytosolic
an
free
secretion
by TRH
Previous transient
of Ca 2+ channels
may
low-threshold
channels
Ca 2+
(6-8),
as well
studies
not
of
thyrotropin-releasing
regulating
([Ca2+]i),
as inhibition
which
the
sustained
results
in
insus-
of voltage-dependent
K+
(4,5).
(T-type)but
the neuropeptide
mechanism
concentration
(i-3),
in GH 3 cells
whole-cell
by
important
of
in GH 3 cells and the
demonstrated
long-lasting transient
the
high-threshold
high-threshold
presence
of
(L-type)-,
(N)-type.
T-
*To whom correspondence should be addressed. Abbreviations used; GTPTS, guanosine 5'-0-3-thiotriphosphate;^GTP, guanosine te-~GDP~S, guanosine 5'-0-2-thiodiphosphate; [CaZ+]$, cytosolic free Ca2 concentration; T current, transient low-threshold Ca2 current; L current, long-lasting high-threshold Ca2+ current.
~
529
0006-291 X/92 $4.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 187, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and L- channels function very d i f f e r e n t l y in the conversion of t i a l s to i n t r a c e l l u l a r calcium transients, (9).
The individual roles of
Ca2+ channel
action poten-
as evidenced by McCobb and Beam
the two d i s t i n c t types
of voltage-dependent
in the specialized hormonal function in GH3 c e l l s remain to be
determined. We have recently suggested that the sustained elevation of [Ca2+]i t r i g gered by TRH in GH3 c e l l s might be due to r e p e t i t i v e openings of T-type Ca2+ channels causing a series of transient Ca2+ influx largely through T-channels (10),
as a result of the enhancement of action potential f i r i n g over a pro-
longed period induced by TRH e f f e c t in GH3 c e l l s (4, 11-14; for a review, see 15).
Also, TRH inhibited L-type Ca2+ current has been c l e a r l y demonstrated by
Levitan and Kramer (8) and Kramer et al. patch clamp technique. through T-type
(16), using the whole-cell perforated
This r e s u l t f u r t h e r suggests that
Ca2+ i n f l u x
Ca2+ channels is responsible for the sustained increase of
[Ca2~] i . In t h i s w h o l e - c e l l voltage-clamp study, we concentrated on examining whether TRH modulates the T-type and
L-type currents in GH3 c e l l s ,
through
the activation of a G protein, which may regulate the frequency of Ca2+ action p o t e n t i a l s , thereby controlling the level of the sustained
[Ca2+]i in GH3
cells.
MATERIALS AND I4ETIIODS Cell culture A cloned line of rat pituitary tumor cells, GII 3 cells, was purchased from American Type Culture Collection (Rockville, MD) through Dainihon-Seiyaku (Oosaka, Japan), and the cells were maintained in Ham's F-IO medium supplemented with 15% horse serum, 2.5% fetal bovine serum under 5% C02/ air at 37 °C (17). The medium contained 50 units/ ml penicillin and 50 g/ ml streptomycin. Cells were plated onto glass coverslips (15 mm diameter) and electrophysiological experiments were performed 2-7 days later. Physiological recording The whole-cell patch-clamp technique (18) was used to examine cells in a bath perfused (i ml/min) at room temperature. The patch electrode resistance ranged from 3 to 8 MD when filled with internal solutions. A model S-3666 patch clamp whole cell clamp amplifier (Nihon Kohden, Tokyo) with a type P-10G probe (feedback resistor: i0 GD) was used. Voltage pulses and currents were sampled online at 200 ~sec intervals with a computer (VM-2, NEC, Japan). Whole cell current measured under voltage clamp conditions was sampled after filtered (+3 db at 3 kHz). The stimulation rate was 0.5 Hz, Linear leak and capacitance currents were subtracted digitally. Ca 2+ currents were recorded with a pipete filled with an intracellular solution containing 145 mM Cs aspartate, 5 mM EGTA A 2 mM Mg-ATP, i0 mM HEPES (pH 7.4); cells were continuously perfused with CaZ+-TEA saline consisting of 2 mM CaCl 2, 138 mM TEA-C1, 2 mM MgCl 2, 20 mM HEPES (pH 7.3 was adjusted by NaOH). Tetrodotoxin (TTX) at a concentration of 500 nM was added to suppress currents through Na + channels. TRH (Sigma) in distilled water was dissolved in the perfusing saline. Nonhydrolyzable GTP analog, guanosine 5'-O-3-thiotriphosphate (GTPTS; Boehringer-Mannheim), an activator of G protein (19), was added to the patch pipet solution at a concentration of 20 ~M in Figs. 1 and 3. In Fig. 4B, GTPTS was added to the patch pipete solution at 50 ~M. In some experiments, hydrolyzable guanosine 5'-triphosphate (GTP, BoehringerMannheim) was added to the patch pipete solution at 20 ~M. Nonhydrolyzab]e
530
Vol. 187, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
GDP a n a l o g , g u a n o s i n e 5 ' - O - 3 - t h i o t r i p h o s p h a t e (GDPflS; B o e h r i n g e r - M a n n h e i m ) , an i n a c t i v a t e r o f G p r o t e i n (19), was added to t h e p a t c h p i p e t s o l u t i o n a t a concentration o f 100 ZM i n F i g . 2. S o l u t i o n c h a n g e s i n t h e c h a m b e r w e r e c o m p l e t e d w i t h i n 3 min. O th e r c h e m i c a l s were from Wako Pure Chem. (Oosaka, Japan).
RESULTS T- a n d L - c h a n n e l s
F i g u r e 1A ( c o n t r o l mY,
depolarizing
above
test
each t r a c e s )
traces)
tionally
L-type
msec t e s t
pulses
associated
(B),
(for
an example,
-20 and -10 mV inactivation
s e e t h e arrow heads
relations
(C) and T c u r r e n t
To i s o l a t e
current
slow
rapid inactivation
components were p r e s u m a b l y f u l l y
current-voltage
L current
circles).
pulses at
showing v e r y
o f -80 indicated with
produced
during
addi-
maintained
The a m p l i t u d e of L - c u r r e n t was measured a t t h e end o f 160
time the T-current the
T - t y p e c u r r e n t s showing
and t e s t
currents
from a h o l d i n g p o t e n t i a l
t o between -50 and -30 mV ( p o t e n t i a l
induced
depolarization.
shows t h a t ,
pulses
maintained depolarization,
i n GH3 c e l l s .
the
experiments of Figs. at a concentration
(D),
amplitude
(C) was s u b t r a c t e d
the
measured
peak
in
amplitude
GTPTS
of
1A),
(D),
at
which
In F i g .
(= T + L)
control
of T c u r r e n t
from t h e
1 and 3,
depict
in Fig.
inactivated.
current
solution
the
( o p en
amplitude
peak c u r r e n t
was added t o t h e p a t c h
(B).
of L In
pipet
o f 20 zM, a l t h o u g h we c o u l d n o t know a c t u a l
1,
the
solution
concentrations
o f GTP7 S r e n d e r e d w i t h i n c e l l s . Effects
o f TRH {500 n~) on T- and L - c u r r e n t s .
F i g u r e 1A (TRH t r a c e s ) T current
amplitude
duced by TRH appreciable
shows t h a t TRH (500 nM) d r a m a t i c a l l y
i n GH3 c e l l s .
was s e e n o v e r
A significant
test
potential
In c o n t r a s t
to t h e i n c r e a s e
1A (TRH t r a c e s )
illustrates
amplitude
current,
as
amplitude
of L c u r r e n t
decrease
of L in
the
that
1C; s o l i d
shows
decrease
that
both this
were a p p r e c i a b l y Figs.
reversed
1C and 1D),
" r u n down" effect
at
to
test
pulses
by TRH
and
the
the
at
of r e c o v e r y ,
the
- 1 0 mV.
The
test
potentials
1A ( R e c o v e r y t r a c e s ) increase
in T c u r r e n t
( s e e a l s o open t r i a n g l e s
t h e d e c r e a s e of L c u r r e n t
extent
but
rather
in
was n o t c a u s e d by by a d e p r e s s i v e
o f TRH.
S u p e r i m p o s e d Ca 2+ c u r r e n t
traces
in F i g .
2 were measured
in the
i n c l u d i n g GDP a n a l o g GDP~S (100 ZM) i n t r o d u c e d w i t h a p a t c h p i p e t , tivator
Figure
decreased
-20 and
Figure
in L c u r r e n t
that
of
pro-
1D; s o l i d
of T c u r r e n t ,
was seen
circles).
when TRH was washed out
indicating
least
in t h e a m p l i t u d e
the
-10 mV, w i t h no
of about -60 mV ( F i g .
TRH (500 nM) d r a m a t i c a l l y
shown a t
e x c e e d i n g -20 mV ( F i g .
of T c u r r e n t
r an g e of -50 t o
change i n t h e t h r e s h o l d p o t e n t i a l
circles).
increase
increased
of G p r o t e i n
no s i g n i f i c a n t
(19).
changes
in
In F i g . the
2B, t h e a d d i t i o n
T currents
531
cell
an i n a c -
o f TRH (500 nM) p r o d u c e d
(compare B w i t h
A),
although
some
V o l . 1 8 7 , N o . 1, 1 9 9 2
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
A -50
-40
-30
-20
T
-10
T
-5O
-40
-30
-20
-10
-50
-40
-30
-20
-10
Control
TRH
Recovery
B
C mV 20
-89_, .~ -4.0 ,
~
D
- 8 9 . . . , .. - ~ o
~.
mV o 29
-80
,.~ - 4 0
mV i
20 I
10(
pA -300
~
~00
pA
500
pA
F i g . 1. TRIt (500 nM) i n c r e a s e d T - t y p e Ca 2+ c h a n n e l c u r r e n t , b u t r e d u c e d Lt y p e Ca 2+ c h a n n e l c u r r e n t . Currents were recorded under whole-cell v o l t a g e - c l a m p i n GH3 c e l l s . I n (A), Ca 2+ c u r r e n t t r a c e s were o b t a i n e d j u s t before (Control traces), d u r i n g a p p l i c a t i o n of 500 nM TRH (TRH t r a c e s ) and f o l l o w i n g r e c o v e r y o f 5 min ( R e c o v e r y t r a c e s ) , t o t h e same GH3 c e l l . From a holding potential of -80 mV, d e p o l a r i z i n g p u l s e s (160 msec d u r a t i o n ) t o b e tween -50 and -30 mV, t h e p o t e n t i a l i n d i c a t e d above each t r a c e s , i n d u c e d Tt y p e c u r r e n t , and t e s t p u l s e s a t -20 and -10 mV i n (A) and -20 and +20 mV i n (B) p r o d u c e d L - t y p e c u r r e n t , t o g e t h e r with T-type current. The a m p l i t u d e o f L - t y p e c u r r e n t was m e a s u r e d a t t h e end o f 160 msec t e s t p u l s e s ( s e e t h e a r r o w heads in Control traces). The a s s o c i a t e d c u r r e n t - v o l t a g e r e l a t i o n s d e p i c t t h e peak (= T - t y p e + L - t y p e ) c u r r e n t (B), L - t y p e c u r r e n t (C) and T - t y p e c u r r e n t (D), m e a s u r e d i n c o n t r o l s o l u t i o n (open c i r c l e s ) , i n t h e p r e s e n c e o f 500 nM TRH ( s o l i d circles), and f o l l o w i n g r e c o v e r y a t 5 min ( o p e n t r i a n g l e s ) . C u r r e n t t r a c e s h a v e been c o r r e c t e d f o r l i n e a r l e a k and c a p a c i t a n c e c u r r e n t i n t h l s and s u c c e e d i n g f i g u r e s . I n f i g u r e s 1 and 3, G T P r S was added t o t h e p a t c h p i p e t s o l u t i o n a t a c o n c e n t r a t i o n o f 20 zM, a l t h o u g h we c o u l d n o t know the actual concentrations o f GTPTS w i t h i n c e l l s . Throughout this e x R e r i m e n t , GH3 c e l l s were b a t h e d in N a + - f r e e , Ca2+-TEA s o l u t i o n t o i s o l a t e Ca c u r r e n t s , and TTX was added t o s u p p r e s s c u r r e n t t h r o u g h Na + c h a n n e l s .
Z+
increases in the L currents were observed (arrow),
i n d i c a t i n g t h a t the cyto-
s o l i c GDP~S markedly blocked the e f f e c t of TRH (500 nM) to produce both an increase of T current and a decrease of L current as seen in Fig. corresponds to the control experiment of Fig. i . 532
1.
This
Vol. 187, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A
B
F i g . 2. C y t o s o l i c GDPflS, an i n a c t i v a t o r of G p r o t e i n , b l o c k e d t h e e f f e c t of TRH as s e e n in Fig. 1. GDPBS (100 ZM) was r e n d e r e d w i t h i n t h e c e l l v i a a p a t c h p i p e t in (A) and (B). Superimposed Ca2+ c u r r e n t t r a c e s in (A) and (B) were evoked w i t h t e s t p u l s e s (160 msec d u r a t i o n ) spaced by 10 mV t o between -SO and -20 mY, and +10 mV ( a r r o w s ) from a h o l d i n g p o t e n t i a l o f -80 mY. Note t h a t i n t h e p r e s e n c e o f t h e c y t o s o l i c GDPflS, T c u r r e n t s in c o n t r o l t r a c e s (A) r e m a i n e d l a r g e l y u n c h a n g e d by t h e a d d i t i o n o f SO0 nM TRH (B), a l t h o u g h some i n c r e a s e in the L - c u r r e n t was o b s e r v e d (B, a r r o w ) . V e r t i c a l and h o r i z o n t a l c a l i b r a t i o n b a r s r e p r e s e n t 50 pA and 40 msec, r e s p e c t i v e l y .
Effect In Fig.
3,
superimposed (arrows), currents
of a lowered concentration control on c u r r e n t
with
o f - 8 0 mV.
traces
t a k e n 30 s e e a f t e r
pulse
T currents
increases
by
in
marked w i t h
(Fig.
1).
S.E.M.;
N=6)
at
The the
of T current
the
test
increase test
pulses
at
-20
of
-40
threshold
-30
to and
traces -30
10
o f 5 nM TRtt show
mY a n d mY,
shown
pure T
L currents
from
a
holding
o f TRft (5 nM) p r o d u c e d s i g n i f similar was
to those by
- 3 0 mY. at
are
the application
-50
T current
potential
by TRH (5 nM) o c c u r r e d
v -50
of
of T current,
- 4 0 a n d - 1 0 mY, w i t h no c h a n g e i n t h e
,
range
of
open triangles,
Control current
k reduced concentration
in the amplitude
nM TRH
crease
traces
evoked by t e s t
potential icant
current
m e a s u r e d f r o m t h e same c e l l .
together
o f T~I (5 nM) on T c u r r e n t s .
test
35 The
potentials
potential
-20
i n d u c e d b y 500
± 6.0%
(mean
appreciable in the
of about
range of
- 6 0 mY and
+10 -
_
~I00 pA 50 ms Fig. 3.
A lowered c o n c e n t r a t i o n of TRH (S nM) markedly i n c r e a s e d t h e a m p l i tude of t h e T c u r r e n t , whereas no a p p r e c i a b l e change o f t h e L c u r r e n t was seen. C o n t r o l c u r r e n t t r a c e s marked with open t r i a n g l e s a r e shown s u p e r i m p o s e d on c u r r e n t t r a c e s taken 30 sec a f t e r the a p p l i c a t i o n of TRH ( a r r o w s ) , in t h e same c e l l . From a h o l d i n g p o t e n t i a l o f - 8 0 mV, t e s t p u l s e s to between -SO and -30 mV as i n d i c a t e d above each t r a c e induced T-type c u r r e n t , and t e s t p u l s e s a t -20 and 10 mV produced both L-type c u r r e n t and T - t y p e c u r rent. Six of 7 c e l l s examined showed s i g n i f i c a n t i n c r e a s e s o f t h e T c u r r e n t s by 35 + 6.0% (mean ± S.E.M.; N=6) at the test potential of -30 mV i n r e s p o n s e t o TRH a p p l i c a t i o n . 533
±
in-
Vol. 187, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A
B
F i g . 4. I n t r a c e l l u l a r l y a p p l i e d GTPTS with a p a t c h p i p e t c o n t a i n i n g 50 ~M GTPrS induced b o t h an i n c r e a s e of T c u r r e n t and a d e c r e a s e o f L c u r r e n t in (B}, t h r o u g h t h e a c t i v a t i o n of a G p r o t e i n , and hence mimicked t h e e f f e c t s of e x t r a c e l l u l a r l y a p p l i e d TRH (500 nM) shown in Fig. 1. F a m i l i e s of s u p e r i m p o s e d Ca2+ c u r r e n t t r a c e s were o b t a i n e d from a c e l l w i t h o u t GTPTS (A) and a c e l l w i t h GTP~S ( B ) . No TRH was p r e s e n t e x t r a c e l l u l a r l y . C u r r e n t s were evoked w i t h t e s t p u l s e s (160 msec d u r a t i o n ) spaced by 10 mV to between -50 and +10 mV f o r (A) and (B) from a h o l d i n g p o t e n t i a l of -80 mV. Note t h a t the Ca 2+ c h a n n e l c u r r e n t was l a r g e l y i n a c t i v a t i n g T c u r r e n t i n (B). (A) and (B) show d a t a from d i f f e r e n t c e l l s . The mean r a t i o o f t h e a m p l i t u d e s of T c u r r e n t measured a t a t e s t p o t e n t i a l of -20 mV r e l a t i v e t o t h e a m p l i t u d e s of L c u r r e n t a t 10 mV was 2.4 (N=3) in a c e l l with 50 Z M GTP7 S, in c o n t r a s t to t h e r e d u c e d mean r a t i o of 1,0 in a c e l l w i t h o u t GTPTS (N=4, d a t a not shown). V e r t i c a l and h o r i z o n t a l c a l i b r a t i o n b a r s r e p r e s e n t 50 pA and 40 msec, r e s p e c t i v e l y , f o r (A) and (B). A broken l i n e in (B) i n d i c a t e s 0 c u r r e n t l e v e l .
the
peak
position
concentration current,
of
of
about
as s e e n at
a test
When h y d r o l y z a b l e GTP~S,
rents
5 min a f t e r
within
potential
~iEicked
from a cell
respectively.
The
of
produced both
current
- 8 0 mV.
not
applied
test
traces
internally
with
of
were
elicited
increments
a patch
lowered of
pipet
of non-
increase
of T
- 3 0 mY.
by
with
test
Ca 2+
z M),
in
the
from a holding
TRH, 50 ZM GTP7 S
of L current
potentials
(50
potentials
(160 msec d u r a t i o n )
of both
concentration
current
GTP 7 S
of
(Fig.
currents GTPTS
was f o u n d t o m i m i c t h e
effects
by externally
TRH
applied
4B), (N=3,
(50
ZM)
on t h e
T- a n d L - t y p e
Ca 2+ c u r r e n t s
induced
These suggest
that
o f TRH (500 nM) i s m e d i a t e d b y a G p r o t e i n .
the effect
L
on t h e T c u r -
This of
superimposed
and a d e c r e a s e
elevated
instead
effect
the absence of extracellular
the
this
amplitude
cells,
similar
GTP7 S and a c e l l
of T current
Hence,
the
o f T~I on T- a n d L - c u r r e n t s .
change in the threshold
shown).
within
potential
representative without
Despite
an i n c r e a s e
w i t h no s i g n i f i c a n t
However, in
o f t h e membrane s e a l . the
the effects
r a n g e o f - 5 0 t o +10 mV i n 10 mV potential
shown). a change
o f 10 mV.
the rapture
4A a n d 4B s h o w
obtained
not
induce
zM) was r e n d e r e d
was b y 27 + 2.0% (N=3) a t
Figures
data
to
TRH (5 nM) a p p e a r e d t o g i v e
GTP7 S (50 ~ )
traces
mV ( d a t a
GTP (20
hydrolyzable
current
-20
TRH (5 nM) f a i l e d
(500
nM}.
DISCUSSION The p r e s e n t significantly
experiments
revealed
augments T-current
for
amplitude, 534
the
first
time
that
TRH (500
through the activation
riM)
of a G pro-
Vol. 187, No. 1, 1992
tein,
in
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
GH3 c e l l s
especially
under
significant
whole-cell
such as
the
enhancement o f t h e f r e q u e n c y of Ca 2+ a c t i o n p o t e n t i a l s
is
increasing
and s u s t a i n i n g
secretion current
a secreting
cell
The i n c r e a s e
we o b s e r v e d i n t h i s
potential
frequencies
s t u d y may r e s u l t
i n GH3 c e l l s ,
d e p r e s s i o n o f K+ c u r r e n t s by TRH ( 4 , 5 ) . mimicked by i n t r a c e l l u l a r l y a p p l i e d GDP~S,
fact
is
i n which
a prerequisite
for
i n t o n i c hormone
in t h e a m p l i t u d e
of T
t h e enhancement o f Ca 2+
possibly
This e f f e c t
concomitant
with
the
o f TRtt on T c u r r e n t
was
a p p l i e d GTP 7 S and was b l o c k e d by i n t r a c e l l u l a r l y
suggesting
d u c t i o n mechanism t h a t
in
This
Gtt3 c e l l ,
t h e l e v e l of [Ca2+]i, which r e s u l t s
( f o r a r e v i e w s e e 15).
that
action
for
voltage-clamp conditions.
that
links
a G protein
TRH r e c e p t o r
is
i n v o l v e d in the
activation
signal
trans-
to the modulation
of T-
t y p e Ca 2+ c h a n n e l s . The i n c r e a s e reversible riM).
in T-current
amplitude
This
T-current
reduction
in L - c u r r e n t
amplitude,
makes i t
prolonged enhanced current.
is
possible
Ca 2+ l e v e l
action potentials [Ca2+]i
is
that
the
and
channel activity
Kramer
(8)
i n which t h e v e s i c l e
retains
indicating
cell
of
or o u t - s i d e
have a p p a r e n t l y
(4,20).
The p r e s e n t
configuration
have is
the
tained
result
using patch pipet
increase
frequencies relatively
largely influx
T is
the accumulated frequency
an a c c u r a t e of
in
during the
calcium
increased
of
regulation
Ca 2+ e n t r y
largely
u n i f o r m and i n s e n s i t i v e
recently
shown t h a t inhibited
named t h e
"perforated
out
patches
that
TRH has
L currents
successfully solution
supplemented
vesicle
method",
in
an o u t -
L-channel conductance; In c o n v e n t i o n a l
compo-
Ca 2+ c h a n n e l s
in conventional
by TRH (500 nM), with
the
whole-
cytoplasmic
on L t y p e
recorded
inhibited
Ca 2÷
decreased the probability
in which c r u c i a l no e f f e c t
L-type
by TRtt (100 nM),
ion channels
t h a t TRH r e v e r s i b l y single
recorded L cur-
single
dramatically
c y t o p l a s m and c o n t a i n s
been l o s t ,
may be
of
but t h a t
c h a n n e l c o n d u c t a n c e i s 32 pS i n 110 mM b ar i u m .
recordings
nents
cell
amount
Furthermore,
o f a L c h a n n e l b e i n g open w i t h o u t a f f e c t i n g single
the
i n d u c e d by TRH i s
findings using whole-cell
clamp c o n f i g u r a t i o n
s i d e out o r i e n t a t i o n ,
to
TRH (500
waveform (9).
from GH3 c e l l s
u s i n g a new p a t c h
net
because
whose amount may be
in a c t i o n p o t e n t i a l
Levitan
firing
by c h a n g i n g
In accordance with the present rents,
contrast
t h e Ca 2÷ c h a n n e l c u r r e n t
as s u g g e s t e d by (16).
through T channels,
in
in response to
of L - t y p e c u r r e n t ,
increased
c o u l d be p o s s i b l e
to alteration
that
Ca 2+ a c t i o n p o t e n t i a l
Hence i t
intracellular
amplitude
likely
reduced because of the i n h i b i t i o n
of
was f o u n d t o be a c c o m p a n i e d by a
d e c r e a s e i n L - t y p e Ca 2+ c u r r e n t a m p l i t u d e
GTPTS
wholewas
(20
obZM).
ACKNOWLEDGr~ENTS
We t h a n k D r s . H. R o b i n s o n and C . - F , Wu f o r r e a d i n g t h e m a n u s c r i p t . T h i s work was s u p p o r t e d by a G r a n t - i n - A i d (03680223) f o r S c i e n t i f i c Research t o N.S. from t h e M i n i s t r y o f E d u c a t i o n , S c i e n c e , and C u l t u r e o f J a p a n . 535
Vol. 187, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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