348
Intra- and Extracellular Actions of Proton on
the Calcium Current of Isolated Guinea Pig
Ventricular Cells
H. IRISAWA AND R. SATO
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External or internal application of proton on the calcium current was investigated using the whole-cell
patch clamp technique in single ventricular cells of the guinea pig. The effect of add pH. on the
calcium current depended on the strength of the buffer in the internal solution. In the presence of 5
mM HEPES in the pipette, external acidification depressed calcium current with the hah* maximum
inhibition of pH, 5.5. When the intracellular pH was buffered strongly to pH 7.2 by 50 mM HEPES
within the pipette solution, half maximum inhibition of the calcium current was shifted to the acidic
side markedly. When acid was applied hi the patch electrode, calcium current was hardly affected by
the pH values down to 6.0 in the pipette solution but was depressed by approximately half on further
acidification to pH 4.5. When the Na + -H + exchange system was blocked by superfusing the cell with
either amiloride or Na+-deficient solutions (Tris substituted), calcium current was decreased by half
at a pipette pH of around'6.5 and was completely and irreversibly blocked at pH^ 6.0. From the above
results, we concluded that the calcium current of the ventricular cell is much more sensitive to
intracellular protons than extracellular ones. (Circulation Research 1986; 59:348-355)
I
T is well known that the calcium current (icJ is
depressed when an excess of protons is applied
either extracellularly '~3 or intracellularly*7 in cardiac muscle. The external and internal actions of protons on ic,, however, have not been analyzed separately since external acid is likely to lower the intracellular
pH (pH,) by an influx of protons across the cell membrane.8 With the acidic pH,, on the other hand, protons
must be expelled by the Na + -H + exchange mechanism7910 (for review, see Aronson"). Measurement of
the pH effect on i^, however, should provide basic
information for clarifying the pathogenesis of low pH
in cardiac muscle since a low pH is considered to be
one of the major causes of disturbance of the mechanical and electrical activities of the heart during myocardial ischemia.!2
In the present study, we demonstrate that the effects
of internal and external application of protons are actually modulated by the movement of protons across the
membrane and that the decreasing effect of protons on
ic is more potent at the inner side than the outer side of
the membrane.
Materials and Methods
Voltage Clamp of Single Myocytes
Single ventricular cells of the guinea pig were obtained by the enzymatic dissociation method as described previously.71314 The whole-cell membrane
currents were recorded by a patch clamp method."
From the National Institute for Physiological Sciences, Okazaki,
Japan.
Supported in part by a grant from the Ministry of Education,
Science and Culture and the Ministry of Health and Welfare, Japan.
Address for reprints: Hiroshi Irisawa, National Institute for Physiological Sciences, Myodaiji-cho, Okazaki 444. Japan.
Received December 6, 1985; accepted May 29, 1986.
Patch electrodes having an inner diameter of approximately 3-4 /-im were fabricated from glass capillaries
(Mertex, Mercer Glass Works, N. Y.). Each electrode
was heat-polished at the tip and was filled with an
internal solution of a given pH. The electrode resistance ranged between 0.6 and 1.3 Mft. The liquid
junction potential between the normal Tyrode solution
and the pipette internal solution was about - 13 mV,
for which measurements of the membrane potentials
were compensated as described by Hagiwara and Ohmori.16 Under an inverted microscope (x 400, Nikon
TMD), the electrode was pressed against the cell surface near the center of a single cell. A seal resistance of
10-100 Gfi was established between the electrode tip
and the membrane by applying a suction of —0.3 to
- 0 . 5 kPa. After allowing the contaminating Ca2+ inside the electrode tip to be buffered by the EGTA of the
pipette solution for several minutes, the patch membrane was ruptured by briefly applying a stronger suction. The rupture was indicated by a sudden appearance of large capacitive currents. The membrane
current was initially outward at the holding potential of
— 30 mV but shifted progressively in an inward direction as shown in Figure 1A. The time course of the
current inward shift may reflect the diffusion of Cs + rich pipette solution into the cell. The half time of the
shift was 2.1 ± 0.8 minutes (n = 10). We used data
from experiments in which the holding current shifted
inward, with a smooth sigmoidal time course (Figure
1 A) and discarded those with a decay time longer than
5 minutes.
The pipettes were filled with a given acidic internal
solution from the beginning in most experiments.
When secondary effects of preloading the acid solution
into the pipette were anticipated, we changed the control pipette solution (pH^ 7.2) to an acidic one during
the voltage clamp, using the pipette perfusion device
Irisawa and Sato
Proton on Calcium Current in Cardiac Cell
349
described elsewhere.717 Briefly, a tapered polyethylene tube was inserted into the patch pipette and various
test solutions were perfused through this tube under a
pressure of - 0 . 3 to - 0 . 5 kPs applied to the pipette.
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FIGURE 1. A. Current changes accompanying membrane disruption and replacement of normal Tyrode with K+ -free Tyrode
solution using the pipette perfusion method. The internal solution was Cs aspartate K+-free solution. At the filled arrow, the
membrane patch was disrupted. The thickened current trace
indicates the current in response to a 1 mV test pulse (duration,
30 msec; frequency, 3.3 Hz; holding potential, —30mV). At the
open arrow, the K*-free Tyrode was superfused. Time scale, 1
minute. The I-V relations were obtained before and after K+free Tyrode superfusion. B. l-V relationship after K*-free
Tyrode superfusion. Filled circles indicate the peak iCa at various test pulses and the open circles indicate the current at 330
msec after onset of the clamp pulse. The inset panel shows a
semilogarithmic plot of the amplitude ofiCa, which can be divided into a fast and a slow component. Holding potential, —30
mV; test pulse, JO mV. C. Original current traces before (1
and 2) and after (3 and 4) K* -free Tyrode superfusion. The
numerals in C correspond to those in A. In 1 and 3, the test
pulse was -40 mV; in 2 and 4, it was +10 mV. The inward
rectifier K+ current was almost abolished and iCa became larger as the extracellular K+ was removed.
TABLE 1.
Solutions
The compositions of the main external and internal
solutions are listed in Table 1. The extracellular pH
(pHJ was changed by titrating the external solution
with HC1. The pH of the internal solutions was adjusted with HEPES-KOH or HEPES-CsOH. PiperazineN,N'bis(2-ethane sulfonic acid), 1.5 Na (PIPES), or 2(N morpholine) ethane sulfonic acid (MES) was
employed instead of HEPES between pH 6.8 and 6.5.
The free Ca concentration of the pipette solution was
higher than pCa 8, which was calculated on the basis of
the apparent stability constants for calcium-EGTA,
calcium-ATP, magnesium-EGTA, and magnesiumATP, which were given by the program 1 provided by
Fabiato and Fabiato.18 Amiloride hydrochloride dihydrate was generously provided by Merck Sharp &
Dohme Research Lab., N.J.
All experiments were performed at 35-36° C. The
results are expressed as the mean ± SD.
Results
Isolation of i^
The membrane was held at — 30 mV and a depolarizing pulse of 300 msec was given to activate i^. To
minimize the outward current, K+ was totally removed
from both the bath and pipette solutions and the cells
were dialyzed internally with 150 mM Cs + . 1920 The
inward rectifier K current was almost completely abolished under these conditions, thus facilitating the measurement of jc,. The original current traces before and
after K+-free Tyrode superfusion are shown in Figure
Composition of Solutions
External solutions:
Control
Tyrode solution
Na-free K-free
(Tris substitution)
Na-free K-free
(TMA substitution)
Na-free K-free
NaCl
136.5
KCl
5.4
CaCl2
1.8
MgCl2
0.5
HEPES
5.0
Glucose
5.5
TrisCl
0
TMACl
0
LiCl
0
0
0
1.8
0.5
5.0
5.5
136.5
0
0
0
0
1.8
0.5
5.0
5.5
0
136.5
0
0
0
1.8
0.5
5.0
5.5
0
0
136.5
Internal solutions:
KH2PO4
MgCl2
CsOH
CsCl
Aspartate
20.0
1.0
1.0
Control
130.0
130.0
Internal solution
1.0
1.0
50 HEPES
20.0
130.0
85.0
Internal solution*
3.0
0
0
42.0
126.0
42EGTA
*42 EGTA solution also contains 20 mM TEA. All concentrations are in mM.
K2ATP
5.0
MgATP
0
EGTA
5.0
HEPES
5.0
5.0
0
5.0
50.0
10.0
42.0
5.0
0
350
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1C. The membrane resistance at the holding potential
was 1.3 Gfi (Figure IB). The magnitude of i^ was
measured as the difference between the peak and the
value at 330 msec from the onset of the clamp pulse.
The maximum i^ was defined as the largest ^ in the
current-voltage (I-V) relations. The potential of the
maximum i^ was defined as the peak potential. In an
example of Figure IB, it was at 10 mV. The amplitude
of maximum i^ was 0.95 ± 0.3 nA (n = 20) and the
estimated current density was 16 /i.A/cm \ assuming
the surface area of the cell to be 6,000 /xm2. When the
magnitude of i a during decay with respect to the
steady-state current level was plotted against time on a
semilogarithmic scale, it could be separated into fast
and slow components. The slow component was fitted
to an exponential time course, and the fast component
was then computed by subtracting the slow component
from the original current trace (Figure IB, inset). The
time constants of the fast (Tf) and the slow ( T J components were 11.3 and 76.4 msec, respectively, at the
peak potential. The membrane current was stable for at
least 30 minutes, and we usually measured the effect of
pH on i^ during this period.
Effects of Extracellular Acidification on iCa
When the pH of the external solution (pHJ was
lowered, [^ was depressed in a manner depending on
the concentration of the pH buffer within the internal
solution. We employed a low (5 mM, Figure 2A) or a
high (50 mM, Figure 2B) HEPES buffer to control the
pH of the pipette solution (pH^) at 7.2. In the presence
of 5 mM HEPES in the pipette, the control I-V relation
was measured at pHo 7.4 (o), and then the pH0 was
lowered to 6.0. The amplitude of the peak i a began to
decrease within 30 seconds after switching on the low
pH perfusate and reached a new quasi-steady level in 3
minutes. The maximum i^ decreased by 47% and the
peak potential shifted along the voltage axis in the
positive direction by about 10 mV during pH0 6.0
perfusion (A). The peak potential of i,-, was —5.1 ±
3.6 mV (n = 10) at pH0 7.4, 3.0 ± 4.6 (n = 7) at pH0
6, 10.0 ± 5.8 (n = 5) at pH0 5 and 14.0 ± 4.2 mV (n
= 2) at pHo 4.5, respectively. This positive shift of the
peak potential could be explained by a change in the
surface potential. The relation between the peak potential and pH0 is shown in "Discussion." The steady-state
current measured at 330 msec into the clamp pulse was
unaffected (Figure 2A). The i^ decay time course was
not significantly changed by the acidic solutions at the
peak potentials.
When 50 mM HEPES was used in the pipette solution, the depressing effect on i^ of the external acidification was reduced. The peak amplitude of i^ was not
reduced for the initial 2 minutes, and it took more than
3 minutes for a sizable effect to develop. In 8 cells, the
pH0 6 solution decreased the maximum i^ in the I-V
relations by 18.5 ± 6.1% (n = 8) in the presence of 50
mM HEPES in contrast to 44.0 ± 13.8% (n = 10)
with 5 mM HEPES. Thus, the more acidic solution
seems to be required to depress iCl significantly, and so
pH0 5.0 was tested. Figure 2B gives an example of the
Circulation Research
-50
0
Vol 59, No 3, September 1986
<|
***
1
/
-l-5nA
6
mV
y5
0-
pHo
FIGURE 2. Effect of decreasing extracellular pH on iCa. A.
I-V relationships before (circles) and after (triangles) superfusion ofpHo 6 solution. • , • indicate the peak current and o, A
indicate the currents at 330 msec after onset of the clamp pulse.
The inset gives the original current traces before (*) and 3
minutes after (A) superfusion of acid solution. The current
trace before the acid superfusion was obtained at a pulse of —3
mV and that after the acid was at +7 mV. The dotted line
indicates the zero current level, and the holding potential was
-43 mV. pH^ was adjusted to 7.2 with 5 mM HEPES. B.
I-V relations before (*, o) and after (A, A) superfusion ofpHo
5 solution. pH^ was adjusted to 7.2 with 50 mM HEPES. The
test pulse before acid was — 7 mV and that after acid was +13
mV. The holding potential was —43 mV. The decay time constants, yand xt of the control iCa were 9.8 ±2.5 msec and 69.2
± 19.4msec(n = 16) at the peak potential (— 3 mV). At pH06,
xf was 9.9 ± 3.6 msec (n = 13) and T, was 78.0 ± 18.1 msec
(n = 13) at the peak potential (+ 7 mV) atSmM HEPES. At the
internal solution of 50 mM HEPES, xfwas 11.0 ±2.3 msec (n
= I l)andx, was59.6 ± 10.7msec (n = 11) at -7mV, in the
control; and at the lower pHo, XfWasll.8 ± 1.4msec(n = 11)
and x, was 68.6 ± 10.3 msec (n = 11) at the peak potential.
The difference was also statistically insignificant, when measured at the peak potential. We compared these time courses
assuming the differences of the peak potential was due to the
presence of surface charge. C. Shift of peak iCa plotted against
pH0. The continuous line is the curve predicted from Equation 2
in "Discussion". The observed maximum slope of the curve in
the presence of 1.8 mM Ca is 11 mV/unit pH0 change and
corresponds to a charge separation of 20 A.
I-V relations before and during superfusion of pH0 5.0
solution in the presence of 50 mM HEPES in the internal solution. Although the positive shift of the peak
potential became larger, i.e., 18 mV in B compared to
about 10 mV in A, the reduction of i^ by pH0 5.0 was
only 22% in this case. This result strongly indicates
that the depressing effect of external acid in 5 mM
HEPES internal solution was largely due to protons
entering the cell. The decay time course of i,^ was not
significantly changed at the peak potentials.
Irisawa and Sato
Proton on Calcium Current in Cardiac Cell
Recovery of i a was always seen when pH0 was
returned to 7.4 from 6.0. The time course of the recovery was not monotonic. iCa increased progressively to a
degree that even exceeded the original amplitude before returning to the control level. Even in an extreme
example of pH0 4, where i a was abolished within 2
minutes, it recovered almost completely within 2 minutes after returning to pHo 7.4 (Figure 3).
The relations between pH0 and the decrease in i,^ at
5 mM and 50 mM HEPES in the pipette solution were
compared in 37 cells. The sigmoidal curve was calculated by the empirical equation
y =
+
(1)
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where ymu is the maximum amplitude; x is the concentration of proton; kw is the concentration of proton that
gives half maximal inhibition of i,^. At 50 mM HEPES
in the pipette, the half maximum inhibition of i a was
obtained at pHo 5.0 in contrast to pHo 5.5 at 5 mM
HEPES. The above findings indicate that perfusion of
the acid solution acidify the cell interior, and the degree of internal acidification is smaller when 50 mM
HEPES than when 5 mM HEPES is in the pipette
solution.
This assumption was supported by using pHpip 8.0
pip
instead of p H ^ 7.2 internal solution. When the pipette
solution was changed from pHpip 7.2 to pHL 8.0 by
using the pipette perfusion method while perfusing pH0
6 solution, the amplitude of i a increased progressively
by about 20%. i c , was then depressed with a very slow
time course (Figure 4; • ) . This observation was in
sharp contrast to the finding that i^ began to decrease
within 30 seconds and reached a new steady level
within 2 minutes when p H ^ was set at 7.2 with 5 mM
HEPES buffer (Figure 4; o). Under the condition of
pHpjp 8 and pH0 5.5, the I-V relations indicate that the
peak potential was at 10 mV and the decrease in i^ was
FIOURE 3. Relation between amplitude of peak iCa and external pH. The amplitude of iCa (ordinate) was normalized and
plotted against pH0 (abscissa). Open circles were obtained with
5 mM HEPES in the internal solution, and filled circles were
obtained with 50 mM HEPES in the internal solution. The pH0,
which gave half maximum inhibition ofiCa on the 50 mM HEPES
curve, was 5.0 and the corresponding pHo on the 5 mM HEPES
curve was 5.5. The correlation coefficient was 0.9 for the 50
mM HEPES (n = 20) and 0.8 for 5 mM HEPES (n = 24).
351
t t t r m
*
100
?
C
<»
f
/
pHf*
r
50
i
i
i
»^»
i
i
i
i
i
i
Time (rrtn)
i
i
i
K)
FIOURE 4. A. Effects of lowering pHo on iCa with a pH^ of
8.0 and 7.2. The amplitude of iCa was normalized and was
plotted against time in minutes. The amplitude ofiCa was 0.8 nA
at zero time. *'s show the amplitude ofiCa on superfusion ofpHo
6.0 solution with pHpjp 8.0 and O's indicate the data obtained
with pHplp 7.2. The holding potential was —30 mV, and test
clamp pulses of + 10 mV were applied repetitiousty. The pipette
perfusion method was employed. Internal solution was the control internal solution (Table 1) with 5 mM HEPES or MES
buffer and control Tyrode solution was superfused. The first
downward arrow indicates the moment when the bath solution
was changed to the pH0 6.0, and the upward arrows indicate
washout with normal Tyrode solution, for both pHpip 8.0 and
7.2. Note the very rapid decrease in iCa with the pH^ 7.2
solution. Inset shows the original current traces, where pHo was
lowered to 6.0 at pHplp 8.0. The holding potential was —30 mV
and the command pulse was 10 mV. Arrows indicate the peak iCa
afterpHo 6.0perfusion. Dotted line gives the zero current level.
25%, which was far smaller than that observed at p H ^
7.2. This result indiates that it took longer time before
reaching low enough p ^ for the proton influx across
the cell membrane, since the cell interior was pretreated with alkaline buffer solution.
From the above findings it can be concluded that the
external application of protons acidifies the cell interior and affects i,^ and that it decreases the negative
charge on the surface membrane.
Effects of Intracellular Acidification on iCa in the
Presence of Amiloride
We found previously that i,^ was depressed by the
application of pH^ 6.0 internal solution only when
Na + was depleted from the bath solution.7 This observation led us to assume that protons diffused into the
cell from the pipette may be extruded through the cell
membrane by the Na-H exchange mechanism." If the
Na-H exchange system is involved, the application of
amiloride, an inhibitor of Na-H exchange, may cause
an accumulation of protons within the cell.
After recording stable iCl for 5 to 10 minutes with
pH^p 6.8 solution, 1 mM amiloride was applied in the
bath. The peak amplitude of i^ decreased progressively with time, and it took 6 minutes before i^ was
reduced by approximately 60% of the control (Figure
5). No significant changes were observed in the threshold and the peak potentials of i^ (Figure 5-1) as well as
352
Circulation Research
1nA
Control
A3
-33
8 mln amllorld*
•27
.
—* CONTROL
» o 5 mm
-3
R«oov«ry
'-3
-=sr
OOmsec
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FIOURE 5. £#i»cr of internal acidification (pHptp 6.7) in the
presence of ] mM amiloride on the membrane current. The left
panel shows the original current traces in the control (top panel), 6 minutes after 1 mM amiloride superfusion (middle panel)
and recovery (bottompanel). The zero current level is indicated
by the horizontal bar at the right side of each trace. Numerals
indicate command potentials in millivolts. The holding potential
was —43 mV. The right panel shows the I-V relations before
(A), 3 minutes (o) and 6 minutes (a) after superfusion of 1 mM
amiloride. The upper panel (1) gives the peak currents and the
lower panel (2) gives the currents at 330 msec after onset of the
clamp pulses. The time constant of inactivation, y was 10.5 ±
1.4 msec (n = 5) in the control, and it was 9.0 ± 1.9 msec (n =
5) atpHpip 6.8 ~ 6.6 after amiloride superfusion. The difference
was thus statistically insignificant, x, was also unchanged (62.9
± 8.9 msec in the control and65.3 ± 10.8 msec (n = 11) with
amiloride).
in the steady state current (Figure 5-2). The time constant of decay at 10 mV was not changed significantly.
The depression of i^ was reversible. When internal
solutions more acidic than p H ^ 6.6 were used, i^ was
reduced more quickly by a greater extent. It reached a
new steady level within 3 minutes, and recovery was
not complete on washing out the test solution. We
applied 1 and 3 mM amiloride in 3 other cells and
found no qualitative difference between these two concentrations, suggesting that the effect of amiloride was
saturated at 1 mM. Superfusion of 1 mM amiloride
exerted no significant effect on the i^ at p H ^ 7.2.
These findings support the view that amiloride blocked
the Na-H exchange mechanism and caused an accumulation of protons within the cell.
The amplitudes of i^ at various p H ^ solutions were
normalized with respect to the control value (pH^ 7.2)
measured before the application of amiloride and plotted against p H ^ (Figure 6, • ) . A significant decrease
in i^ was observed already at pH^, 6.8, and the half
maximum effect was obtained at p H ^ 6.6. The decrease in the maximum i^ was compared before and
after changing the pipette pH solution in the absence
(Figure 6, o) or presence of amiloride (Figure 6, • ) , in
a separate series of experiments. The decrease of i^
was not observed until pHpip of 5.5, and it was only
approximately 50% even at a p H ^ 4.5. These results
strongly suggest that the influx of protons from the
Vol 59, No 3, September 1986
pipette is almost counterbalanced at a pH higher than
5.5 by an efflux through the Na-H exchange mechanism under normal conditions.
Effects of Intracellular Acidification on iCa Under
Na+-Free Conditions
The involvement of the N a + - H + exchange mechanism in preventing the cell from internal acidification
was further examined by depleting the external Na + .
The ic, was at first obtained in normal Tyrode solution
with the acidic p H ^ solution and then the external Na +
was replaced with Tris totally. The amplitude of i^.
was markedly reduced (over 50%) when pH^, was
lower than 6.8. At p H ^ 6.0, i ^ was irreversibly abolished in most experiments. There was a small difference in the i^ decay time course: xf was 12.0 ± 2.0
msec in the control, and 8.0 ± 2.0 msec (n - 5) after
Na + removal. This difference was not observed in the
presence of external Na. It is known that absence of
Na + by itself does not change the inactivation time
course,20 and so it may be an indirect effect of Na
removal.
Removal of Na,, has the following possible complications. Total removal of Na0 decreases the pH,,9
which in turn increases the Ca2+,.2122 The faster decay
time course would be explained by assuming an increase of Ca,. In addition, the continuous extrusion of
protons in exchange for Na + might cause Na + accumulation within the cell during the period that the control
and subsequent removal of the external Na + reversed
the Na + gradient across the membrane and caused an
increase in Ca 2+ influx through the Na-Ca exchange
mechanism. Thus, under the Na-free condition, both
the block of Na-H exchange and the increase of Ca,
50-
FIGURE 6. Effect of various internal pH values on the peak iCa
in the absence (O) or presence C) of 1 mM amiloride. The
amplitude of iCa at low pHpip (ordinate) was normalized and is
plotted against various pH^ (abscissa). Without application of
amiloride, iCa was not significantly affected by the internal
acidification (kiA waspHpip 4.5; correlation coefficient was 0.9,
n = 16). In the presence of amiloride, kt/i shifted to pH^ 6.6
(correlation coefficient was 0.9, n = 22). Under this condition,
therefore iCa became very sensitive to internal proton. The continuous curves were drawn by the same method as in Figure 3.
Irisawa and Sato
Proton on Calcium Current in Cardiac Cell
Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017
occur within the cell, which may complicate the results.
In order to avoid such complications, the pipette was
perfused at first with prLp 7.2 solution while superfusing Na + -depleted Tyrode solution, and then p H ^ was
lowered. This method should minimize the increase of
Ca, due to Na-Ca exchange antiport because the exchange must be negligible under the Na-free condition
in the pipette. Furthermore, we employed 42 mM
EGTA internal solution to avoid alteration of Caj in 8
experiments.
When the p H ^ was changed from 7.2 to 6.8 under
these conditions, the i c , amplitude was appreciably
reduced (36.3 ± 7.6%, n = 4). The threshold and the
peak potentials were unchanged (Figure 7A). The difference in time course of decay at the peak potential
was not significant. The relation between the amplitude of ^ and various pH^p values in Na + -free Tyrode
solution (Figure 7B) indicated that i^ was directly
affected by p H p and the depression reached almost
maximum at p H ^ 6.0. The half maximum inhibition
of i a was observed at pW^ ^ y.l. In three experiments,
we substituted Na + with TMA totally and obtained
similar results. These findings suggest that i a of the
ventricular cell is much more sensitive to intracellular
protons than extracellular protons. Results of experiments using 42 mM EGTA in the pipette solution as
well as the fact that the similar pHp, titration curve
between Na-free (Tris substituted) and amiloride (Figures 6 and 7) suggest that our result does not contain an
effect of intracellular Ca increase.
When we substituted all the Na + with Li + , the decrease in i^ at pHpl 6.8 was 4.6 ± 3.8% (n = 5), at
pH p l p 6.5itwas21.6 ± 8.5% (n = 7) and at prT 6.2 it
was 27.0 ± 3.5% (n = 2). The depression or l^ was
smaller than that obtained when substituting Na + with
-50
353
Tris or TMA, suggesting that Li + in the absence of
Na + appears to exchange for H + at a rate lower than
that of Na + .
For additional confirmation, we compared the effects between reducing only pH, and both pH, and pH0
to 6.0. In the former case, very little effect was observed on the amplitude of i^ (Figure 6, o), but in the
latter case when both pHp,p and pH0 was 6.0, i a was
abolished in three experiments. This observation indicated that in the latter condition the Na-H exchange
was reduced and pH, became close to pHpip.
Discussion
The present experiments revealed that the depressing effects of protons on i^ is far more potent from
inside than from outside in isolated guinea pig ventricular cells. The ^ is markedly reduced with a relatively
small change in internal pH (pHpip 6.8) when Na-H
exchange was blocked. The half maximum inhibition
of i^ was obtained approximately at p H ^ of 6.6. The
half maximum inhibition by pH0 is 5.0-5.5. Deitmer
and Ellis found that the intracellular pH changes by
0.23 units/pH0 in Purkinje fibers.8 If it is also true in
our preparation, superfusion of pH0 5.5-6.0, which
induced 50% inhibition, reduces pH, from 7.2 to
6.8-6.9. These values coincide well with Deitmer and
Ellis's calculation, and it is possible to reduce i^ intracellularly.
Our conclusion is broadly consistent with results
obtained in other tissues or in other classes of channels. In squid axon, the Na + current is more sensitive
to changes in internal than in external pH.232* In Paramecium, internal acidification depressed i^, whereas
external proton exerted little effect.25 Protons are also
known to block the internal site of the channel for the
inward rectifier K + current,2627 delayed rectifier K +
FICJURE 7. A. Effect ofpHplp 6.8 under superfusion of
Na-free K-free Tyrode solution. The pipette contained 42
mM EGTA. The original tracings show the superimposed
current traces at pHpip 7.2 (o) and at pH^ 6.8 (•) in the
right panel and in the left panel show the recovery. The
iCa was completely recovered when pH^ 7.2 was reperfused in the pipette. The dotted line indicates the zero
current level. The holding potential was —30mV and a
clamp pulse to 0 mV was applied. The time courses of
inactivation, x/andxI were 20.1 ± 2.7 and 69.9 ± 6.5
msec (n = 8) in pHplp 7.2, respectively. Those in pHplp
6.8 were 20.7 ± 7.1 and 68.0 ± 9.2 msec (n = 8),
respectively. In the I-V relations, solid curves indicate
the initial current level at pHpip 7.2 (o) and at pH^ 6.8
(•). The dotted curves give those at 330 msec after onset
of the clamp pulse. B. The dose-response relation between the iCa amplitude (ordinate) and various internal
pH values (abscissa) under the condition of Na-free (Tris
substituted) Tyrode solution. The amplitude of iCa was
normalized and is plotted against pHpip. The continuous
curve was drawn by the same method as in Figure 3. The
pHpip which gave half maximum inhibition was 6.7, and
the correlation coefficient was 0.8 (n — 16).
Circulation Research
354
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current28 and acetylcholine-induced K + current.29
These findings, together with our results, suggest that
the ionic channels are generally suppressed by protons
more effectively from the inside of the membrane than
from the outside.
The threshold potential, the peak potential, and the
time course of decay were unchanged by lowering
p H ^ and only the amplitude of i^ was decreased.
These results suggest that proton affects the limiting
conductance of iCl and probably does not affect the
kinetics of the channel, although further experiments
are needed to confirm these notions.
The presence of the Na-H exchange mechanism in
heart cells has been reported by various workers.7"10
Amiloride is known to be a potent inhibitor of Na-H
exchange, and its maximal effect was obtained at 10"4
M.3031 Since the pH titration curve was the same in
both Na + -free (Tris substituted) and 1 mM amiloride
Tyrode solution, we assumed that 1 mM amiloride
might block the Na-H exchange almost completely. It
has been demonstrated that Li + inhibits Na-H exchange in membrane vesicles of the renal microvillus.31 Aickin and Thomas found that Li + was unable to
substitute for Na + in mouse skeletal muscle,30 but in
sheep Purkinje fibers Ellis and MacLeod found Li +
was able to exchange for H + . 9 In crayfish neurons,.
Na+-free (Li + ) Ringer solution was about 80 to 95% as
effective in blocking pH: recovery.32 In the present
study, in Na + -free solution where all the Na + was
substituted by Li + , the reduction of i^ was always
smaller than those observed with other Na + substitutes
such as Tris or TMA. Li + may thus partly substitute
Na + in the Na-H exchange system.
Figure 2C illustrates the relation between the shifted
peak potential and the external pH. The smooth curve
in Figure 2C was drawn by using the Gouy-Chapman
equation3334:
a = (1/G) [1,0, (exp- z i F ^T -
1)]*
(2)
where a is the presumed surface charge density
(0.0025 e~'/A 2 ), G is a constant of 270 at a temperature of 10° C, R, F, and T have their usual meanings, Z,
is 1 for proton, C, is the external ionic concentration
including proton, and vy0 is the outer surface potential.
It should be noted that the shift of the peak potential
was almost saturated at around pH0 7.4. Lowering the
pH0 below 6 shifted the peak potential in the positive
direction. It is concluded that externally applied protons probably affected the negative surface charge of
the membrane and produced the shift in the peak i^.
The charge density may not be as large as in other
cells.33"33 We estimated the charge separation in the
ventricular cell using the Gouy-Chapman equation and
obtained a value of 20 A, which was sparser than in
other cells (9 A in egg membrane34). The direct effect
of the surface charge on the amplitude of ^ is, however, negligibly small. Our findings disagree with the
results described by Kohlhardt et al, 2 who did not observe a shift of the peak i^ by external acid application.
We did not record a shift of the peak i,^ during intracellular acidification. This may be due either to the small-
Vol 59, No 3, September 1986
er density of the negative charges at the inner site of the
membrane or to the small pH range tested in the case of
the pipette application.
Acknowledgments
The authors wish to thank Profs. H. Ohmori and A. Noma for
critically reading the manuscript. They are also grateful to their
colleagues at the N.I.P.S., Okazaki, and the technical help of M.
Obara and O. Nagata. Thanks are due to Profs. H. Okumura and H.
Hayakawa, Nippon Medical School, for providing R.S. with the
opportunity of conducting joint research at the N.I.P.S., Okazaki.
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• single ventricular cell • calcium current • proton effect • Na-H exchange
• intra- and extracellular effect of proton
KEY WORDS
Intra- and extracellular actions of proton on the calcium current of isolated guinea pig
ventricular cells.
H Irisawa and R Sato
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Circ Res. 1986;59:348-355
doi: 10.1161/01.RES.59.3.348
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