1095
ATP Directly Affects Junctional Conductance
Between Paired Ventricular Myocytes Isolated
From Guinea Pig Heart
Hiroki Sugiura, Junji Toyama, Naoya Tsuboi, Kaichiro Kamiya, and Itsuo Kodama
Downloaded from http://circres.ahajournals.org/ by guest on October 2, 2016
Effects of ATP on junctional conductance (gj) were investigated in paired ventricular myocytes
isolated from guinea pig hearts. One cell of the pair was voltage-clamped with a single-patch
pipette, and gj was measured after the perforation of the nonjunctional membrane of the
partner cell. The current-voltage relation of gj was linear between -30 and +30 mV. The control
gj at 5.0 mM ATP in 88 pairs of cells ranged from 100 to 1,055 nS (average, 268 nS). ATP within
the range from 0.1 to 5.0 mM increased gj in a dose-dependent manner. The Hill coefficient was
2.6, and the half-maximum effective concentration of ATP was 0.68 mM. Adenylylimidodiphosphate (2 mM) caused a transient increase in gj in the presence of 0.5 mM ATP, but forskolin
(30 ,uM), cyclic AMP (50 ELM), catalytic subunit of cyclic AMP-dependent protein kinase (1
,uM), and ADP (10 mM) had no significant effect on gj. The temperature coefficient of gj in the
presence of 5.0 mM ATP was 1.29. These findings suggest that gj in paired ventricular myocytes
is directly regulated by ATP probably through a specific ligand-receptor interaction between
ATP and gap junctional channel protein. (Circulation Research 1990;66:1095-1102)
P rimary effects of ischemia on cardiac muscle
include decreases of ATP and creatine phosphate within affected fibers. These decreases
result in a depression of active transport systems,
which elevates intracellular concentrations of Na+,
Ca2+, and protons.1 Previous studies2-4 have shown
that electrical coupling was suppressed when energy
metabolism was inhibited by various experimental
conditions, such as hypoxia or ischemia, or in the
presence of metabolic inhibitors. In some cases,
uncoupling effects have been shown to be reversed by
intracellular injection of ATP2 or EDTA3 or by
addition of glucose to the extracellular perfusate.4
Abundant studies indicate that increases in intracellular concentrations of Na+, Ca2 , or protons5-7
cause cell decoupling, and it has been assumed that
uncoupling effects of ischemia are due to changes in
these ions. It has also been reported that in various
tissues junctional conductance (gj) is either increased8-10 or decreased1l-13 as a consequence of
From the Department of Circulation and Respiration, The
Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan.
Supported by Grants-in-Aid for Scientific Research (01624004
and 63480226) from the Ministry of Education, Science and
Culture of Japan.
Address for reprints: Hiroki Sugiura, MD, Department of
Circulation and Respiration, The Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku,
Nagoya 464-01, Japan.
Received August 16, 1989; accepted November 20, 1989.
cyclic AMP (cAMP)-mediated phosphorylation of
the channel protein. Recent advances in cardiac
electrophysiology have proved that some ionic channels in sarcolemmal membrane can be regulated by
ligand-receptor interaction between ATP and channel proteins14 or by phosphorylation of channel
proteins.15
In the present study, we investigated the direct
effect of ATP on cardiac gj by using the whole-cell
clamp of paired ventricular myocytes, one of which
was perforated to allow intracellular diffusion with
extracellular solutions.7 We report here that gj is
strongly affected by ATP from 0.1 to 5.0 mM and that
this effect is direct and, thus, analogous to the effect
reported on other sarcolemmal channels.
Materials and Methods
Preparations
Enzymatic procedure for the isolation of paired
ventricular cells was modified from the method as
described by Mitra and Morad.16 Briefly, guinea pigs
weighing 150-250 g were killed by an overdose of
pentobarbital sodium. The heart was quickly
removed, and the aorta was immediately cannulated
for Langendorff perfusion. Subsequently, the coronary artery was perfused backward with Ca2`-free
Tyrode's solution for 3-4 minutes. Thereafter, the
heart was perfused by 50 ml Ca2+-free Tyrode's
solution containing 50 mg collagenase (type I, Sigma
Chemical, St. Louis, Missouri) and 10 mg protease
1096
Circulation Research Vol 66, No 4, April 1990
TABLE 1. Composition of Internal Solutions
Na2ATP MgCl2 Aspartic acid CsOH EGTA HEPES TEA Na2CRP
5.0
3.0
110
110
4
5
20
Pipette (mM)
5
Test solution(mM)
5.0
1 (control)
3.0
110
110
4
5
20
0
1.5
1.5
110
110
4
2
5
20
0
110
110
4
5
1.0
20
3
1.0
0
0.5
0.5
110
4
110
4
5
20
0
0.1
5
0.1
110
110
4
5
20
0
TEA, tetraethylammonium chloride; CRP, creatine phosphate. pH of internal solutions was adjusted to 7.2 with
CsOH. ATP concentrations in internal test solutions 2-5 are varied to 1.5, 1.0, 0.5, and 0.1 mM, respectively. In each
of the test solutions, the concentration of MgCl2 was varied to keep free-Mg2' concentration less than 0.3 mM.
Downloaded from http://circres.ahajournals.org/ by guest on October 2, 2016
(type XIV, Sigma Chemical) for 3-10 minutes. Then,
the collagenase solution was washed out by perfusing
with high-K' and low-Cl- solution (KB medium).'7
At the end of the above procedure, the ventricle was
cut into pieces and was stored in the KB medium at
40 C before use. Cells were dispersed in the recording
chamber (0.1-0.2 ml) on the stage of an inverted
phase-contrast microscope (Diaphoto-TMD, Nikon,
Japan) and were continuously perfused with normal
Tyrode's solution (1.8 mM Ca2) at a rate of 2-3
ml/min. About 50% of the cells were rod-shaped in
normal Tyrode's solution. Paired cells were distinguished from single cells by the presence of contact
regions between apposed cells.
Solutions
Normal Tyrode's solution consisted of (mM) NaCl
136.9, NaH2P04 0.33, KCI 5.4, CaCl2 1.8, MgC12 0.5,
glucose 5, and HEPES 5; pH was adjusted to 7.4 with
NaOH. Internal solutions are listed in Table 1. Ca2free Tyrode's solution was prepared by omitting CaCi2
from normal Tyrode's solution. Pipette solution was
essentially the same as the internal test solution (test
solution 1) except that creatine phosphate was added
as a reservoir of high-energy phosphates.
Internal test solutions 1-5 were used to perfuse
the perforated cell interior at various ATP concentrations. To keep free-Mg2+ concentration constant
in the internal test solution, the amount of MgCl2 was
varied between 0.1 and 3.0 mM in proportion to ATP
concentrations. Free-Ca2+ and -Mg2+ concentrations
in each of internal test solutions, which had been
estimated with the stability constants for ATP, Ca2+,
Mg2+, and EGTA at a given pH,1819 were limited to
a level less than 1 nM and 0.3 mM, respectively.
Noma and Tsuboi7 showed that these Ca2+ and Mg2+
concentrations had no effects on gj.
Forskolin, cAMP, and adenylylimidodiphosphate
were directly dissolved in the internal test solutions.
ADP was dissolved in the internal test solution
containing 10 ,uM Pl,P5-di(adenosine-5') pentaphosphate (AP5A), a potent inhibitor of adenosine kinase.
Catalytic subunit of cAMP-dependent protein kinase
(c-subunit) was dissolved in the internal test solution
containing 32.4 mM DL-dithiothreitol to prevent the
inactivation of c-subunit. In preliminary experiments,
AP5A and DL-dithiothreitol
had no effect on gj
(results not shown). All experiments were carried out
at 34°-35° C unless otherwise stated.
Electrophysiological Experiments
The method for measuring gj and the intracellular
perfusion technique were essentially similar to those
as described by Noma and Tsuboi7 except that the
whole-cell clamp was carried out by using a singlepatch pipette instead of the double-pipette method.
In brief, one cell of the pair was voltage-clamped with
a giga-sealed patch pipette filled with pipette solution (Table 1). After clamping the membrane potential of a paired cell at approximately 0 mV, bath
solution was switched from normal Tyrode's solution
(1.8 mM Ca'+) to a Cs+-rich and low-Ca'+ internal
test solution (test solution 1). Subsequently, the
nonjunctional membrane of the partner cell was
perforated by crushing the tip of the other pipette
against the cell. This procedure allowed access of
bath solution to the junctional channel of the perforated cell. Under this condition, current from the
clamped cell flows through two pathways. One is
through the nonjunctional membrane of the clamped
cell to the ground. The other is through the gap
junction and the interior of the perforated cell to the
ground. The equivalent circuit after perforation of
the nonjunctional membrane of the partner cell is
illustrated in Figure 1. The value of input conductance (gi) through the electrode after cell membrane
perforation was given by
gi = gml + gj x (gm2 + gs)/(gj +gm2 + gs)
where g, is the conductance through the hole made
in the partner cell and g., and g.2 are the nonjunctional membrane conductances of the clamped cell
and the perforated cell, respectively. Since g, is
much greater than gj and g,2 the approximate value
of gj was obtained by
gj=gi-gmi
The value of gm, was approximated as half of the
total nonjunctional membrane conductance of a
paired cell (gml+gm2), which had been measured
before perforation of the nonjunctional membrane of
the partner cell. Series resistance of the electrode
Sugiura et al Effects of ATP on Gap Junction
were considered to be negligible, because the negative pressure of pipette and the input resistance
under control condition at 5.0 mM ATP were kept
constant. Paired cells having gjs of larger than 100 nS
were used in our experiments. When membrane leak
conductance increased noticeably within 3 minutes
after cell membrane perforation, the cells were discarded. Ramp command potential between -30 and
+30 mV with a duration of 2.5 seconds was applied
to the clamped cell every 6 seconds. gi was calculated
from the slope conductance of the current output.
Values were presented in mean+SEM unless otherwise stated. Statistical analysis was performed by
Student's paired t test, and values of p<0.05 were
considered to indicate a significant difference.
V
rplp
gml
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Results
Control g,
Figure 2 illustrates changes of gi after the perforation of the nonjunctional membrane of the partner
cell. Current output in response to the command
potential of +30 mV was increased suddenly after
the perforation (Figure 2A). The experiment was
carried out at 5.0 mM ATP in the bath solution.
Therefore, the average value of gi was increased from
47±3 to 292±15 nS (n=88) (Figure 2B). The
current-voltage relations before and after the perforation were linear over the range from -30 to +30
mV (Figure 2C), indicating no voltage dependency of
junctional as well as nonjunctional membrane conductances under such Cs+-rich and low-Ca21 conditions.
The control g. was calculated to be from 100 to 1,055
nS (average, 268±15 nS).
Effects of ATP Reduction on gj
We measured gi with internal test solutions containing various ATP concentrations (0.1-5.0 mM).
gap junction
FIGURE 1. Diagrams showing voltage-clamp circuit and the
equivalent circuit of the open-cell system. One cell of the pair
was voltage-clamped with a patch pipette. I, current; V,
voltage; s.r.c., series resistance compensation; rpip, resistance
of the pipette; gml and gm2, the conductances of the clamped
cell (1) and the perforated cell (2), respectively; gj, the
conductance of the junctional membrane; gs, the conductance
of the hole made in the partner cell. Diagram of the paired cell
is shown on the right.
(1-5 Mfl) was always 1% or less of input resistance
(the parallel sum of the seal and nonjunctional
membrane resistances) and was compensated by the
series resistance compensation circuit in the amplifier. Access resistance of the electrode produced by
cellular materials plugging the pipette was not compensated. However, changes of access resistance
A
llllll
11 llllll
C
+30mV
l l
I
ll
l llll
I (nA)
20
nA
a
-~
-50
ro-
0
U)
c
--20
A
1
min
b
-30
1 min
B
1097
FIGURE 2. Tracings and
graphs showing change of input
conductance (gi) after cell membrane perforation. Panel A:
Original ramp voltage-clamp
pulse between -30 and +30 mV
and current output after the perforation. Closed triangle (A)
indicates the time of cell membrane perforation. Panel B:
Change of gi after cell membrane perforation. gi was sudV(mV) denly increased from 14 to 501
nS by the perforation and
'°
became stable in 1 minute.
Panel C: Current (I)-voltage
(V) relation before and after cell
membrane perforation. Tracings
obtained at the times indicated by
'a' and 'b' in panel A are illustrated. I-V relations obtained
before and after the perforation
were linear between -30 and
+30 mV
Circulation Research Vol 66, No 4, April 1990
1098
ATP
(mM)[-
0.5
5
1
5
5
0.1
(3)
100
(22)
300-
000
isvU)
200 -
.
',-0%-~.0
7.
00
(D
0
a
5
ZW
(8)
100-
0-
0
r
5
10
15
ATP Concentration (-log(M))
Time (min)
Downloaded from http://circres.ahajournals.org/ by guest on October 2, 2016
FIGURE 3. Graph showing effects of ATP on junctional
conductance (gj). ATP concentration in the bath solution was
changed from 5.0 to 0.5, 1.0, and 0.1 mM as indicated at the
top. In this experiment, gj in the presence of 5.0 mMATP was
203 nS. When ATP concentration was decreased from 5.0 mM
to 1.0, 0.5, and 0.1 mM, gj decreased to 123, 46, and 34 nS,
respectively. The ATP-dependent change in gj was reversible.
Ordinate shows gj; abscissa shows the time after the initiation
of the whole-cell clamp.
Our preliminary experiments have shown that with
potassium channels blocked by cesium and tetraethylammonium chloride, g,, values of ventricular
myocytes were unaffected by similar changes in ATP
concentrations of the bath solution (results not
shown). Therefore, gj was calculated by assuming that
the initial g,, value was kept constant throughout the
experiment.
Representative results are shown in Figure 3.
When ATP concentration was reduced from control
(5.0 mM) to 1.0, 0.5, or 0.1 mM, gj decreased in a
concentration-dependent manner. Linear currentvoltage relation of gi indicating no voltage dependency of gj was maintained even at the lower ATP
concentrations. At the resumption of control ATP
(5.0 mM), gj returned toward the control level. In 18
of 40 experiments (45%), half or more gj recovery was
not attained at the resumption of 5.0 mM ATP. Such
irreversible data were discarded, and the remaining
22 experiments were used for data analysis. The
irreversible decoupling is most likely explained by
mechanical breakdown of gap junctional structure
due to rigor induced by ATP depletion.
Figure 4 summarizes the results obtained from 22
pairs of myocytes. Values of gj measured at each ATP
concentration were normalized with reference to the
average of those at 5.0 mM ATP before and after the
reduction of ATP. The smooth curve is a least-squares
fit of the Michaelis-Menten equation to the plotted
dose-response data. The Hill coefficient was 2.6, and
the half-maximum effective concentration of ATP was
0.68 mM. The calculated Michaelis-Menten curve
suggests that gj would reach 0 nS at ATP concentrations less than 0.1 mM. However, we were not able to
determine the exact ATP level causing complete cell
FIGURE 4. Graph showing relation between ATP concentration and normalized junctional conductance (Gj). Ordinate
shows the percentage of Gj with reference to the value
measured at 5.0 mMA TP in each experiment. Abscissa shows
ATP concentration in the bath solution in logarithmic scale.
Data are presented as mean ±SEM. Closed circle shows the
control value in the presence of 5.0 mM ATP. Figures in
parentheses are numbers of experiments. The smooth curve is
a least-squares fit of the Michaelis-Menten equation to the
plotted data. The Hill coefficient was 2.6, and the halfmaximum effective concentration ofA TP was calculated to be
0.68 mM
decoupling because most experiments under such
conditions showed irreversible gj reduction.
The above-mentioned results have revealed that gj
is directly regulated by ATP under the condition in
which free-Ca21, -Mg2+, and -proton concentrations
are kept constant. The following experiments were
designed to clarify possible underlying mechanisms
for the regulation.
Effects of Forskolin, cAMP, and c-Subunit on g,
The ATP-dependent change in gj could be explained by phosphorylation of gap junctional channel
proteins through activation of cAMP-dependent protein kinase. If such a mechanism is responsible for
the opening of gap junctional channels, gj would be
increased by stimulation of adenylate cyclase in the
cell membrane or by an elevation of cAMP or
c-subunit in the cytoplasm. This prediction was tested
by applying forskolin (30 ,uM), cAMP (50 ,uM), or
c-subunit (1 ,uM) to internal test solutions containing
5.0 mM or 0.5 mM ATP.
Application of these three substances for 5 minutes in the medium containing 5.0 mM ATP had no
significant effect on gj (Table 2). Figure 5 shows
effects of forskolin (30 ,uM) and cAMP (50 ,uM)
applied for 3 minutes in the low ATP (0.5 mM)
medium. These substances did not reverse gj values,
which had been decreased by the ATP reduction
(Table 3). These findings may indicate that opening
of gap junction by ATP is not mediated by cAMPdependent phosphorylation of channel proteins.
Sugiura et al Effects of ATP on Gap Junction
TABLE 2. Change in Junctional Conductance in the Presence of
5.0 mM ATP
TABLE 3. Change in Junctional Conductance in the Presence of
0.5 mM ATP
gj (nS)
gj (nS)
After Gj (%)
Before
94±5
247±38
233±28
Forskolin (n=5)
97±2
236±27 225±23
Cyclic AMP (n= 13)
89±6
334±62 284±44
c-Subunit (n=7)
99±5
Adenylylimidodiphosphate (n=5) 195±21 192±18
168±14 169±17 102±8
ADP (n=5)
Values are mean±SEM. gj, junctional conductance; Gj, normalized gj; c-subunit, catalytic subunit of cyclic AMP-dependent
protein kinase. Values of Gj were normalized by the value of gj
measured just before the addition of each drug in the presence of
5.0 mM ATP.
Effects of Adenylylimidodiphosphate and ADP on gj
Some sarcolemmal channels have been reported to
be regulated by the ligand-receptor interaction
Downloaded from http://circres.ahajournals.org/ by guest on October 2, 2016
A
ATP(mM) |
5
0.5
l
S
1~~~~~~~~~~~~
130pm forakow
5001
0.-.0-
0
C
c
a
. .......'.
.
0]
O
ATP(mbo)l
5
1
5
10
Q.5
5
I5#.M CAMP
300 1
..~~
U)
C
c
0
15~0
10
5
Before
After
36+16
27+9
Gj (%)
102+29
54±33
Forskolin (n=3)
63+11
42+11
Cyclic AMP (n=9)
Adenylylimidodiphosphate (n=7) 70+ 11 112±22 156+19*
25±6 82+27
42±21
ADP (n=4)
Values are mean±SEM. gj, junctional conductance; Gj, normalized gj. Values of Gj were normalized by the value of gj measured
just before the addition of each drug in the presence of 0.5 mM
ATP.
*Significantly different from the value of gj measured before the
addition of drugs atp<0.05.
between ATP and channel proteins.14 The ATPdependent change in gj might be explained by a
similar mechanism. We tested this alternative possibility by using nonhydrolyzable ATP analogue (adenylylimidodiphosphate) and ADP. Application of
adenylylimidodiphosphate (2 mM) or ADP (10 mM)
to the internal bath solution containing 5.0 mM ATP
did not affect gj (Table 3).
Figure 6 shows experiments in the low (0.5 mM)
ATP medium. Application of adenylylimidodiphosphate (2 mM) caused a partial and transient reversal of
gj that had been decreased by the ATP reduction from
5.0 to 0.5 mM. Peak gj values during adenylylimidodiphosphate application were 56% larger than the
pretreatment level at 0.5 mM ATP (Table 3). However,
the increment of gj was not maintained and dissipated
spontaneously within several minutes (Figure 6A).
Application of ADP (10 mM) at 0.5 mM ATP
did not cause such a partial reversal of gj (Figure
6B, Table 3).
Effects of Temperature on g,
Effects of temperature on gj were examined in the
control (5.0 mM) ATP medium. As the bath temperature was decreased from 350 to 28° and 240 C, gi
decreased in steps, and the change was reversible
(Figure 7). The temperature coefficient (Q1o) in 10
experiments was 1.29.
I
~ ...
~~
,~~~~~~~~~~~
, r
1099
B
0J
Time (min)
FIGURE 5. Panel A: Graph showing effects of forskolin on
junctional conductance (gj). A reduction ofATP from 5.0 to
0.5 mM resulted in a decrease of gj from 452 to 42 nS.
Application of forskolin (30 MM) for 3 minutes caused no
significant change in gj. Panel B: Graph showing effects of
cyclic AMP (cAMP) on gj. A reduction ofATP from 5.0 to 0.5
mM resulted in a decrease ofgj from 262 to 62 nS. Application
of cAMP (50 MM) for 3 minutes caused no significant change
in gj. For both panels, ordinate shows gj, and abscissa shows
the time after the initiation of the whole-cell clamp.
Discussion
In the present experiments, we measured gj of
ventricular cell pairs isolated from adult guinea pig
hearts through the use of a single voltage-clamp
approach. The current-voltage relation of junctional
membrane was linear between -30 mV and +30 mV,
indicating that gj has no voltage dependency under
such Cs+-rich and low-Ca2' conditions. This finding is
in agreement with previous studies on adult ventricular cell pairs of guinea pig or rat.7,20,21 Recently,
Rook et a122 have shown that gj of neonatal rat heart
pairs exhibits marked voltage and time dependency
when cell coupling is inhibited by a decrease of the
number of channels involved. However, the voltage
dependency is not observed until the transjunctional
potential exceeds ±50 mV. Therefore, our data are
1100
.
Circulation Research Vol 66, No 4, April 1990
0.5
5
ATP(mM)
500 l
A
5
| 2mM AMP-PNP
Temp(TC) |35
1\
400
r"**,"%
00
28
1 33.2
24
1
1
0111
0
0
0-~~~~~~~~~
o~~~~~~~~~
\Ito.
A
0
5
10
0-J
1T
Time (min)
ATP(mM) 1
0.5
5
B
_-
A
5
150-1
100-
1.0-
~rlpr.'O..
0.5-
Downloaded from http://circres.ahajournals.org/ by guest on October 2, 2016
Q1o=1.29
50 0
*
0-
.1
6
B
0
0
itw^wo-4c
i0
1'5
Time (min)
FIGURE 6. Panel A: Graph showing effects of adenylylimidodiphosphate (AMP-PNP) on junctional conductance (gj).
A reduction ofA TPfrom 5.0 to 0.5 mM resulted in a decrease
of gj from 432 to 123 nS. Application of 2 mM AMP-PNP
caused a partial and transient reversal of gj from 123 to 192
nS. This reversal was not maintained and dissipated spontaneously during application of AMP-PNP. Panel B: Graph
showing effects ofADP on gj. A reduction ofATP from 5.0 to
0.5 mM caused a decrease of gj from 105 to 9 nS. Application
of 10 mMADP for 3 minutes caused no significant change in
gj. For both panels, ordinate shows gj, and abscissa shows the
time after the initiation of the whole-cell clamp.
also in agreement with Rook et a122 regarding the
voltage range examined.
We estimated gj values by assuming that nonjunctional membrane conductance of the voltageclamped cell (g.,) is one half of the input conductance before perforation of the other cell of the pair
(gm,+gm2). There is no evidence indicating that gm, is
similar to g.2 and that gm, is kept constant throughout
the experiments even after perforation. Accordingly,
such an assumption might introduce variable errors
in estimating the precise amount of gj values. The
potential error is considered to be minimal under the
control condition in which gm, is less than 10% of gi,
but it may increase as gi declines. Although this
would limit the quantitative accuracy, we nevertheless think that it does not invalidate our conclusion.
Our control gj values in ventricular cell pairs
(average, 268 nS) are more or less similar to those
reported by Kameyama (710 nS in guinea pig)23 and
those by Weingart and Maurer (250 nS in guinea pig
0.1
-
25
30
Temperature (C)
35
FIGURE 7. Panel A: Graph showing temperature-dependent
change ofjunctional conductance (gj) in the presence of 5.0
mM ATP. Temperature of the bath solution was changed
within the range from 240 to 350 C, as indicated at the top.
Ordinate shows gj; abscissa shows the time after the initiation
of the whole-cell clamp. In this experiment, gj at 350 C was
294 nS. When temperature of the bath solution was varied to
28', 240, and 33.20 C, gj decreased to 248, 174, and 279 nS,
respectively. Panel B: Graph showing relation of normalized
junctional conductance (Gj) and temperature. Gj, which was
normalized with the value of gj measured at 35° C at 5.0 mM
ATP, was plotted in logarithmic scale against the temperature.
Temperature coefficient (Q10) was 1.29.
and 590 nS in rat).21,24 Noma and Tsuboi7 and
Wittenberg et a125 showed somewhat higher control
values (1,000 nS in guinea pig, 1,250-2,530 nS in rat).
Such discrepancy might be attributed to different
methods for cell isolation and for gj measurement or
to different species employed.
In the present experiments, gj decreased in a
concentration-dependent manner when intracellular
ATP concentration was reduced to less than 1.5 mM
under the condition where free-Ca21, -Mg2+, and
-proton concentrations were kept constant. This
ATP-dependent change in gj was reversible. The
half-maximum effective concentration of ATP was
0.68 mM.
Intracellular ATP concentration of intact cardiac
myocytes under physiological conditions is kept at a
level ranging from 3.0 to 7.5 mM.26-28 Under some
pathological conditions in which energy metabolism
is inhibited, a depletion of creatine phosphate is
followed by a significant decrease in ATP. Recent
studies have shown that intracellular ATP concentration was decreased by 10-90% from control when
Sugiura et al Effects of ATP on Gap Junction
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cardiac muscles were treated by ischemia or hypoxia
for 10-25 minutes.26-28 ATP-dependent active ionic
transport through sarcolemma and through membranes of intracellular organelles is inhibited under
such conditions leading to an accumulation of Ca21
and H' in the cytosol.2-4 Cell decoupling induced by
ischemia or hypoxia has been mainly attributed to an
elevation of these intracellular cations.2-4 The present data suggest that the ATP depletion may also
contribute to cell decoupling.
As to the effect of cAMP on cell coupling, there
are some discrepancies among previous reports. In
horizontal cells of the turtle retina"112 or fish retina,'3
the permeability of gap junction was shown to be
inhibited by an increase of intracellular cAMP. In
contrast, De Mello9 demonstrated that intracellular
injection of cAMP into cardiac myocytes enhanced
cell-to-cell coupling. Burt and Spray'0 have recently
shown the similar improvement of cardiac cell coupling byr an elevation of intracellular cAMP under
low-Ca + conditions. In addition, Wojtczak29 has
shown that cAMP had no effects on cell coupling
under physiological conditions but inhibited cell coupling during calcium-overload conditions, such as
hypoxia.
cAMP is known to affect many aspects of cell
functions including energy metabolism, contraction,
and intracellular ionic concentrations. An elevation
of cAMP in these previous studies may have affected
gj through various secondary changes in such cell
functions. The present results seem to indicate that
cAMP has no direct action on cardiac gj, provided
intracellular ATP, Mg2+, Ca2+, and H+ are kept
constant.
Recently, Pressler and Hathaway30 showed that
the isolated heart gap junctional protein is phosphorylated by cAMP-dependent protein kinase like liver
or lens gap junctional protein.8,3' However, it is not
clear whether phosphorylation of gap junctional protein increases or decreases cell coupling. In the
present study, gj was not affected by cAMP. This may
indicate that the open state of the gap junctional
channel is not maintained by phosphorylation of the
channel protein.
Adenylylimidodiphosphate caused a partial and
transient reversal of gj that had been depressed by
the ATP reduction. Such a transient reversal of gj was
not induced by ADP. These findings indicate that
nonhydrolyzable ATP analogue (adenylylimidodiphosphate), which has a similar molecular structure to ATP, could partially substitute for ATP.
Accordingly, we suggest that gj is maintained by ATP
through the ligand-receptor interaction between
ATP and gap junctional channel protein. However,
the partial reversal of gj by adenylylimidodiphosphate
was not maintained and dissipated spontaneously
within several minutes. Further experimental studies
will be required to clarify the underlying mechanism
for this phenomenon.
Our data showed that Qlo of the gap junction was
1.29, in agreement with experiments on the junction
1101
of earthworm septate median giant axon.32 This Qlo
value is comparable with that of aqueous diffusion of
ions (Q1o= 1.3) and is much lower than that of
enzyme reactions (Q,0=3), such as phosphorylation
regulated by high-energy phosphates.33 Such a low
Qlo value seems to support our proposal that the
regulation of gap junctional channel is mediated by
ligand-receptor interaction between ATP and channel proteins, because it would require minimal
energy consumption.
In summary, ATP regulates cell coupling between
paired ventricular myocytes independently of intracellular free Ca2+, Mg2+, and protons. We suggest
that ATP-dependent change in gj is mediated by the
ligand-receptor interaction between ATP and gap
junctional channel protein.
Acknowledgments
We express our appreciation to Dr. David C.
Spray for his useful suggestions and comments in
preparing this manuscript.
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KEY WORDS * paired ventricular myocytes * junctional
conductance * ATP * ligand-receptor interaction
ATP directly affects junctional conductance between paired ventricular myocytes isolated
from guinea pig heart.
H Sugiura, J Toyama, N Tsuboi, K Kamiya and I Kodama
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Circ Res. 1990;66:1095-1102
doi: 10.1161/01.RES.66.4.1095
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